KR20220031554A - CD33 Targeted Immunotherapy - Google Patents

Abstract

The present disclosure provides improved compositions and CD33 targeting polypeptides for adoptive T cell therapy to treat, prevent, or ameliorate at least one symptom of cancer, infectious disease, autoimmune disease, inflammatory disease, and immunodeficiency, or conditions related thereto. provides

Description

CD33 Targeted Immunotherapy

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is filed under 35 U.S.C. § 119(e) asserts the benefit of Provisional U.S. Patent Application Serial No. 62/898,392, filed September 10, 2019, and Provisional U.S. Provisional Patent Application No. 62/845,304, filed May 8, 2019, each of which is incorporated herein by reference in its entirety.

STATEMENT REGARDING SEQUENCE LISTING

The sequence listing associated with this application is provided in text format instead of a paper copy, and is incorporated herein by reference. The name of the text file containing the sequence listing is BLBD_119_02WO_ST25.txt. The text file created on May 5, 2020 is 302 KB and is submitted electronically through EFS-Web at the same time as the filing of this specification.

technical field

The present disclosure relates to improved adoptive cell therapy directed against CD33. More specifically, the present disclosure relates to chemically regulated signal transduction molecules containing anti-CD33 VHH, chimeric antigen receptors containing anti-CD33 VHH, cells, and related therapeutic methods using the same.

Description of related technologies

The global burden of cancer doubled between 1975 and 2000. Cancer is the second leading cause of morbidity and mortality worldwide, with approximately 14.1 million new cases reported and 8.2 million deaths due to cancer in 2012. The most common cancers are breast cancer, lung and bronchial cancer, prostate cancer, colon and rectal cancer, bladder cancer, melanoma of the skin, non-Hodgkin's lymphoma, thyroid cancer, kidney and kidney cancer, endometrial cancer, leukemia, and pancreatic cancer. The number of new cancer cases is expected to increase to 22 million within the next 20 years.

Adoptive cell therapy is emerging as a powerful paradigm for delivering complex biological signals for cancer treatment. In contrast to small molecule compositions and biological drug compositions, adoptive cell therapy has the potential to perform unique therapeutic tasks due to its myriad sensory and response programs and progressively elaborating mechanisms of gene regulation. Existing methods have mainly focused on scFv-based chimeric antigen receptors (CARs). CAR T cell therapy has achieved limited success due to insufficient CAR expression, in vivo proliferation of CAR T cells, rapid loss of cells after injection, disappointing clinical activity, and antigen escape.

There is a need to retrofit immune effector cells with improved machinery and/or improved CARchitectures for sensing and integrating chemical and/or biological information associated with the local physiological environment.

The present disclosure generally provides a VHH-based dimerization agent modulated immunoreceptor complex (DARIC) directed against CD33 and a VHH-based chimeric antigen receptor (CAR), polynucleotides encoding the same, compositions thereof, and for treating cancer It relates in part to methods of making and using them.

In certain embodiments, the VHH DARIC or VHH CAR binds full length CD33. In certain embodiments, the VHH DARIC or VHH CAR binds to a CD33 splice variant. In certain embodiments, the CD33 splice variant lacks the 124 amino acids encoded by exon 2 of the human CD33 gene (CD33 C2 variant). In certain embodiments, the CD33 splice variant lacks the 54 carboxy-terminal amino acids due to an early translation termination signal resident in exon 7a. In certain embodiments, the CD33 splice variant lacks the 124 amino acids encoded by exon 2 and lacks the 54 carboxy-terminal amino acids due to an early translation termination signal resident in exon 7a.

In certain embodiments, VHH DARIC or VHH CAR binds full-length CD33 and CD33 splice variants.

In various embodiments, the non-native cell comprises a first polypeptide and a second polypeptide, wherein the first polypeptide comprises: an FRB multimerization domain polypeptide or variant thereof; CD8α transmembrane domain or CD4 transmembrane domain; CD137 costimulatory domain; and/or a CD3ζ primary signaling domain, wherein the second polypeptide comprises: an anti-CD33 VHH antibody having the amino acid sequence set forth in any one of SEQ ID NOs: 2-21; FKBP multimerization domain polypeptide or a variant thereof; and a CD4 transmembrane domain or a CD8α transmembrane domain, wherein the bridging factor is associated with and disposed between the multimerization domains of the first and second polypeptides, thereby promoting formation of a polypeptide complex at the non-native cell surface.

In certain embodiments, the anti-CD33 VHH antibody has the amino acid sequence set forth in SEQ ID NO:10.

In certain embodiments, the anti-CD33 VHH antibody has the amino acid sequence set forth in SEQ ID NO:20.

In certain embodiments, the FKBP multimerization domain is FKBP12.

In some embodiments, the FRB polypeptide is FRB T2098L.

In certain embodiments, the bridging factor is selected from the group consisting of: AP21967, sirolimus, everolimus, novolimus, pimecrolimus, ridaforolimus, tacrolimus, temsirolimus, umirolimus and zotarolimus.

In various embodiments, the first polypeptide comprises a signal peptide, a CD8α transmembrane domain; CD137 costimulatory domain; and a CD3ζ primary signaling domain.

In certain embodiments, the second polypeptide comprises a signal peptide and a CD4 transmembrane domain.

In a further embodiment, the second polypeptide comprises a costimulatory domain.

In some embodiments, the costimulatory domain of the second polypeptide is selected from a costimulatory molecule selected from the group consisting of: Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9. , TLR10, caspase recruitment domain family member 11 (CARD11), CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD94, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS) ), DNAX-activating protein 10 (DAP10), T cell lineage member 1 (LAT), leukocyte protein of 76 kD containing SH2 domain (SLP76), T cell receptor associated transmembrane adapter 1 (TRAT1), TNFR2, TNFRS14, TNFRS18, TNRFS25, and the zeta chain of T cell receptor associated protein kinase 70 (ZAP70).

In a further embodiment, the co-stimulatory domain of the second polypeptide is a co-stimulatory domain isolated from OX40 or TNFR2.

In a further embodiment, the second polypeptide comprises an amino acid sequence set forth in any one of SEQ ID NOs: 22-31.

In certain embodiments, the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO:30.

In a preferred embodiment, the first polypeptide comprises the amino acid sequence set forth in SEQ ID NO:82.

In various embodiments, the non-naturally occurring cell comprises a polypeptide complex comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises: an FRB multimerization domain polypeptide or a variant thereof; CD8α transmembrane domain or CD4 transmembrane domain; CD137 costimulatory domain; and/or a CD3ζ primary signaling domain, wherein the second polypeptide comprises: an anti-CD33 VHH antibody having the amino acid sequence set forth in any one of SEQ ID NOs: 2-21; FKBP multimerization domain polypeptide or a variant thereof; and a CD4 transmembrane domain or a CD8α transmembrane domain; and a bridging factor disposed between and associated with the multimerization domains of the first and second polypeptides.

In certain embodiments, the anti-CD33 VHH antibody has the amino acid sequence set forth in SEQ ID NO:10.

In certain embodiments, the anti-CD33 VHH antibody has the amino acid sequence set forth in SEQ ID NO:20.

In certain embodiments, the FKBP multimerization domain is FKBP12.

In certain embodiments, the FRB polypeptide is FRB T2098L.

In some embodiments, the bridging factor is selected from the group consisting of: AP21967, sirolimus, everolimus, novolimus, pimecrolimus, ridaforolimus, tacrolimus, temsirolimus, umirolimus and zotarolimus.

In a further embodiment, the first polypeptide comprises a signal peptide, a CD8α transmembrane domain; CD137 costimulatory domain; and a CD3ζ primary signaling domain.

In certain embodiments, the second polypeptide comprises a signal peptide and a CD4 transmembrane domain.

In some embodiments, the second polypeptide comprises a costimulatory domain.

In various embodiments, the costimulatory domain of the second polypeptide is selected from a costimulatory molecule selected from the group consisting of: Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9 , TLR10, caspase recruitment domain family member 11 (CARD11), CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD94, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS) ), DNAX-activating protein 10 (DAP10), T cell lineage member 1 (LAT), leukocyte protein of 76 kD containing SH2 domain (SLP76), T cell receptor associated transmembrane adapter 1 (TRAT1), TNFR2, TNFRS14, TNFRS18, TNRFS25, and the zeta chain of T cell receptor associated protein kinase 70 (ZAP70).

In a further embodiment, the co-stimulatory domain of the second polypeptide is a co-stimulatory domain isolated from OX40 or TNFR2.

In a further embodiment, the second polypeptide comprises an amino acid sequence set forth in any one of SEQ ID NOs: 22-31.

In certain embodiments, the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO:30.

In a preferred embodiment, the first polypeptide comprises the amino acid sequence set forth in SEQ ID NO:82.

In certain embodiments, the cell is a hematopoietic cell.

In certain embodiments, the cell is a T cell, an αβ T cell, or a γδ T cell.

In a further embodiment, the cell is a CD3+, CD4+, and/or CD8+ cell.

In various embodiments, the cell is an immune effector cell.

In some embodiments, the cell is a cytotoxic T lymphocyte (CTL), a tumor infiltrating lymphocyte (TIL), or a helper T cell.

In a further embodiment, the cell is a natural killer (NK) cell or a natural killer T (NKT) cell.

In various embodiments, the source of cells is peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue derived from the site of infection, ascites, pleural effusion, spleen tissue, or a tumor.

In certain embodiments, the FRB multimer and domain and the FKBP multimerization domain are localized extracellularly when the first polypeptide and the second polypeptide are expressed.

In some embodiments, the fusion polypeptide comprises a first polypeptide and a second polypeptide, wherein the first polypeptide comprises: an FRB multimerization domain polypeptide or variant thereof; CD8α transmembrane domain or CD4 transmembrane domain; CD137 costimulatory domain; and/or a CD3ζ primary signaling domain; a polypeptide cleavage signal, wherein the second polypeptide comprises: an anti-CD33 VHH antibody having the amino acid sequence set forth in any one of SEQ ID NOs: 2-21; FKBP multimerization domain polypeptide or a variant thereof; and a CD4 transmembrane domain or a CD8α transmembrane domain.

In certain embodiments, the anti-CD33 VHH antibody has the amino acid sequence set forth in SEQ ID NO:10.

In certain embodiments, the FKBP multimerization domain is FKBP12.

In certain embodiments, the FRB polypeptide is FRB T2098L.

In some embodiments, the bridging factor is selected from the group consisting of: AP21967, sirolimus, everolimus, novolimus, pimecrolimus, ridaforolimus, tacrolimus, temsirolimus, umirolimus and zotarolimus.

In a further embodiment, the first polypeptide comprises a signal peptide, a CD8α transmembrane domain; CD137 costimulatory domain; and a CD3ζ primary signaling domain.

In certain embodiments, the second polypeptide comprises a signal peptide and a CD4 transmembrane domain.

In certain embodiments, the fusion polypeptide comprises a sequence set forth in any one of SEQ ID NOs: 32-41.

In certain embodiments, the fusion polypeptide comprises the sequence set forth in SEQ ID NO:40.

In a further embodiment, the second polypeptide comprises a costimulatory domain.

In various embodiments, the costimulatory domain of the second polypeptide is selected from a costimulatory molecule selected from the group consisting of: Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9 , TLR10, caspase recruitment domain family member 11 (CARD11), CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD94, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS) ), DNAX-activating protein 10 (DAP10), T cell lineage member 1 (LAT), leukocyte protein of 76 kD containing SH2 domain (SLP76), T cell receptor associated transmembrane adapter 1 (TRAT1), TNFR2, TNFRS14, TNFRS18, TNRFS25, and the zeta chain of T cell receptor associated protein kinase 70 (ZAP70).

In a further embodiment, the co-stimulatory domain of the second polypeptide is a co-stimulatory domain isolated from OX40 or TNFR2.

In certain embodiments, the polypeptide cleavage signal is a viral self-cleaving polypeptide.

In certain embodiments, the polypeptide cleavage signal is a viral self-cleaving 2A polypeptide.

In various embodiments, the polypeptide cleavage signal is a viral self-cleaving polypeptide selected from the group consisting of: foot-and-mouth disease virus (FMDV) (F2A) peptide, equine rhinitis A virus (ERAV) (E2A) peptide, Thosea asigna virus, TaV) (T2A) peptide, porcine Tescovirus-1 (PTV-1) (P2A) peptide, Theilovirus 2A peptide, and encephalomyocarditis ) Virus 2A peptide.

In some embodiments, the fusion polypeptide comprises a sequence set forth in any one of SEQ ID NOs: 42-61.

In some embodiments, the fusion polypeptide comprises a sequence set forth in any one of SEQ ID NOs: 50 or 60.

In a further embodiment, the FRB multimer and domain and the FKBP multimerization domain are localized extracellularly when the first polypeptide and the second polypeptide are expressed.

In various embodiments, the polypeptide complex comprises a first polypeptide and a second polypeptide, wherein the first polypeptide comprises: an FRB multimerization domain polypeptide or variant thereof; CD8α transmembrane domain or CD4 transmembrane domain; CD137 costimulatory domain; and/or a CD3ζ primary signaling domain, wherein the second polypeptide comprises: an anti-CD33 VHH antibody having the amino acid sequence set forth in any one of SEQ ID NOs: 2-21; FKBP multimerization domain polypeptide or a variant thereof; and a CD4 transmembrane domain or a CD8α transmembrane domain; and a bridging factor disposed between and associated with the multimerization domains of the first and second polypeptides.

In certain embodiments, the anti-CD33 VHH antibody has the amino acid sequence set forth in SEQ ID NO:10.

In certain embodiments, the FKBP multimerization domain is FKBP12.

In a further embodiment, the FRB polypeptide is FRB T2098L.

In certain embodiments, the bridging factor is selected from the group consisting of: AP21967, sirolimus, everolimus, novolimus, pimecrolimus, ridaforolimus, tacrolimus, temsirolimus, umirolimus and zotarolimus.

In certain embodiments, the first polypeptide comprises a CD8α transmembrane domain; CD137 costimulatory domain; and a CD3ζ primary signaling domain.

In various embodiments, the second polypeptide comprises a CD4 transmembrane domain.

In a further embodiment, the second polypeptide comprises a costimulatory domain.

In some embodiments, the costimulatory domain of the second polypeptide is selected from a costimulatory molecule selected from the group consisting of: Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9. , TLR10, caspase recruitment domain family member 11 (CARD11), CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD94, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS) ), DNAX-activating protein 10 (DAP10), T cell lineage member 1 (LAT), leukocyte protein of 76 kD containing SH2 domain (SLP76), T cell receptor associated transmembrane adapter 1 (TRAT1), TNFR2, TNFRS14, TNFRS18, TNRFS25, and the zeta chain of T cell receptor associated protein kinase 70 (ZAP70).

In certain embodiments, the co-stimulatory domain of the second polypeptide is a co-stimulatory domain isolated from OX40 or TNFR2.

In certain embodiments, the cell is a hematopoietic cell.

In various embodiments, the cell is a T cell, an αβ T cell, or a γδ T cell.

In various embodiments, the cell is a CD3+, CD4+, and/or CD8+ cell.

In a further embodiment, the cell is an immune effector cell.

In some embodiments, the cell is a cytotoxic T lymphocyte (CTL), a tumor infiltrating lymphocyte (TIL), or a helper T cell.

In certain embodiments, the cell is a natural killer (NK) cell or a natural killer T (NKT) cell.

In a further embodiment, the source of cells is peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue derived from the site of infection, ascites, pleural effusion, spleen tissue, or a tumor.

In a further embodiment, the FRB multimer and domain and the FKBP multimerization domain are localized extracellularly when the first polypeptide and the second polypeptide are expressed.

In a preferred embodiment, the first polypeptide comprises the amino acid sequence set forth in SEQ ID NO:82.

In certain embodiments, the chimeric antigen receptor (CAR) comprises: an anti-CD33 VHH antibody having the amino acid sequence set forth in any one of SEQ ID NOs: 2-21; hinge domain; transmembrane domain; one or more intracellular costimulatory signaling domains; and/or a primary signal transduction domain.

In certain embodiments, the anti-CD33 VHH antibody has the amino acid sequence set forth in SEQ ID NO:10.

In certain embodiments, the anti-CD33 VHH antibody has the amino acid sequence set forth in SEQ ID NO:20.

In various embodiments, the CAR comprises in the 5' to 3' direction: an anti-CD33 VHH antibody having the amino acid sequence set forth in any one of SEQ ID NOs: 2-21; hinge domain; transmembrane domain; one or more intracellular costimulatory signaling domains; and/or a primary signal transduction domain.

In certain embodiments, the anti-CD33 VHH antibody has the amino acid sequence set forth in SEQ ID NO:10.

In certain embodiments, the anti-CD33 VHH antibody has the amino acid sequence set forth in SEQ ID NO:20.

In certain embodiments, the hinge domain and the transmembrane domain are isolated from CD8α, CD27, CD28, CD33, CD37, CD45, CD64, CD71, CD80, CD86, CD 134, CD137, CD152, CD154, AMN, and PD1.

In a further embodiment, the one or more costimulatory signaling domains is isolated from a costimulatory molecule selected from the group consisting of: CD28, CD134, CD137, and CD278.

In certain embodiments, the CAR comprises a CD8α signal peptide, a CD8α hinge and transmembrane domain, a CD134 costimulatory domain, and a CD3ζ primary signaling domain.

In a further embodiment, the CAR comprises the amino acid sequence set forth in any one of SEQ ID NOs: 62-81.

In a further embodiment, the CAR comprises the amino acid sequence set forth in any one of SEQ ID NOs: 70 or 80.

In some embodiments, a polynucleotide encoding a first or second polypeptide, a fusion polypeptide, or a CAR contemplated herein is provided.

In various embodiments, a cDNA encoding a first or second polypeptide, a fusion polypeptide, or a CAR contemplated herein is provided.

In certain embodiments, RNA encoding a first or second polypeptide, a fusion polypeptide, or a CAR contemplated herein is provided.

In a further embodiment, a vector comprising a polynucleotide contemplated herein is provided.

In certain embodiments, the vector is an expression vector.

In certain embodiments, the vector is a transposon.

In a further embodiment, the vector is a piggyBAC transposon or a sleeping beauty transposon.

In certain embodiments, the vector is a viral vector.

In certain embodiments, the vector is an adenoviral vector, an adeno-associated virus (AAV) vector, a herpes virus vector, a vaccinia virus vector, or a retroviral vector.

In a further embodiment, the retroviral vector is a lentiviral vector.

In various embodiments, the lentiviral vector is selected from the group consisting of: human immunodeficiency virus 1 (HIV-1); Human Immunodeficiency Virus 2 (HIV-2), Visna-Maedi Virus (VMV) Virus; caprin arthritis-encephalitis virus (CAEV); Equine Infectious Anemia Virus (EIAV); Feline Immunodeficiency Virus (FIV); bovine immunodeficiency virus (BIV); and simian immunodeficiency virus (SIV).

In various embodiments, a cell comprising a first or second polypeptide, a fusion polypeptide, or a CAR contemplated herein is provided.

In certain embodiments, the cell is a hematopoietic cell.

In certain embodiments, the cell is an immune effector cell.

In various embodiments, the cell is a T cell, an αβ T cell, or a γδ T cell.

In some embodiments, the cell expresses CD3+, CD4+, CD8+, or a combination thereof.

In certain embodiments, the cell is a cytotoxic T lymphocyte (CTL), a tumor infiltrating lymphocyte (TIL), or a helper T cell.

In a further embodiment, the cell is a natural killer (NK) cell or a natural killer T (NKT) cell.

In certain embodiments, the composition comprises a cell contemplated herein.

In certain embodiments, a composition comprises a physiologically acceptable carrier and a cell contemplated herein.

In a further embodiment, a method of treating a subject in need thereof comprises administering to the subject an effective amount of a composition contemplated herein.

In certain embodiments, a method of treating, preventing, or ameliorating at least one symptom of cancer, infectious disease, autoimmune disease, inflammatory disease, and immunodeficiency syndrome, or conditions related thereto, comprises administering to a subject an effective amount of a composition contemplated herein. administering.

In some embodiments, a method of treating a subject with a solid cancer comprises administering to the subject an effective amount of a composition contemplated herein.

In various embodiments, the solid cancer is from the group consisting of lung cancer, liver cancer, stomach cancer, colorectal cancer, head and neck cancer, urothelial cancer, prostate cancer, testicular cancer, endometrial cancer, pancreatic cancer, breast cancer, cervical cancer, ovarian cancer, skin cancer, and melanoma. is chosen

In certain embodiments, a method of treating hematological malignancy comprises administering to a subject an effective amount of a composition contemplated herein.

In various embodiments, the hematologic cancer is leukemia, lymphoma, or multiple myeloma.

In certain embodiments, the hematologic cancer is acute myeloid leukemia (AML).

1A depicts an illustration of a VHH-DARIC polypeptide complex.
1B shows an illustration of the CD33 VHH DARIC architecture.
Figure 2a shows the expression of CD33 VHH1-5 DARIC in transduced T cells as detected by anti-VHH staining (top row) and CD33-Fc binding (bottom row).
Figure 2b shows the expression of CD33 VHH9-10 DARIC in transduced T cells when detected by CD33-Fc binding.
3A shows the phenotype of T cells transduced with CD33 VHH1-5 DARIC or control.
Figure 3b shows the phenotype of T cells transduced with CD33 VHH9-10 DARIC or control.
4A shows IFNγ secretion from CD33 VHH1-5 DARIC cells or control cells co-cultured with CD33 ⁺ THP-1 cells at an E:T ratio of 1:1 in the presence or absence of AP21967 for 24 hours.
4B shows IFNγ secretion from CD33 VHH9-10 DARIC cells or control cells co-cultured with CD33 ⁺ THP-1 cells at an E:T ratio of 1:1 in the presence or absence of AP21967 for 24 hours.
4C shows CD33 VHH9 co-cultured with modified 293T cells expressing either full-length CD33 (CD33M) or CD33 splice variants (CD33m, C2) at an E:T ratio of 1:1 in the presence or absence of AP21967 for 24 h. Show IFNγ secretion from ~10 DARIC cells or control cells.
5A shows CD33 expression in MV4-11 cells, MV4-11 cells engineered to knockout the CD33 gene (CD33-KO cells), and unstained controls.
5B shows IFNγ secretion from anti-CD33 VHH9 DARIC T cells or UDT T cells co-cultured with MV4-11 cells or CD33-KO cells at an E:T ratio of 1:1 in the presence or absence of AP21967 for 24 hours. show
5C shows 1:1 with MV4-11 cells (left panel) or CD33-KO cells engineered to express CD33m splice variants (CD33-KO-C2 cells; right panel) in the presence or absence of AP21967 for 24 h. IFNγ secretion is shown from UTD T cells, anti-CD33 CAR T cells, or anti-CD33 VHH DARIC T cells co-cultured at an E:T ratio of .
6 shows IFNγ secretion from anti-CD33 VHH DARIC T cells co-cultured with CD33 ⁺ THP-1 cells at an E:T ratio of 1:1 in the presence or absence of AP21967 for 24 hours.
7 shows anti-CD33 VHH DARIC T cells co-cultured with CD33 ^neg 293 T cells transfected with different amounts of mRNA encoding CD33 at an E:T ratio of 1:1 in the presence or absence of AP21967 for 24 hours. shows IFNγ secretion from
Figure 8a shows immunodeficient NSG mice inoculated with HL60 AML tumor cells expressing a luciferase reporter and treated with UTD T cells or anti-CD33 VHH DARIC T cells in the absence of rapamycin, 10 days after inoculation (day 0). shows the measured tumor growth as a function of luminescence.
FIG. 8B shows immunodeficient NSGs inoculated with HL60 AML tumor cells expressing a luciferase reporter and 10 days post inoculation (day 0) treated with UTD T cells or anti-CD33 VHH DARIC T cells and 1 mg/kg rapamycin. Tumor growth measured in mice is shown as a function of luminescence.

Brief description of sequence identifiers

SEQ ID NO: 1 shows the amino acid sequence for full-length human CD33.

SEQ ID NOs: 2-21 set forth the amino acid sequence for the anti- CD33 VHH domain.

SEQ ID NOs: 22-31 set forth the amino acid sequence for the anti-CD33 VHH DARIC binding component.

SEQ ID NOs: 32-41 set forth the amino acid sequences for anti-CD33 VHH DARIC fusion proteins.

SEQ ID NOs: 42-51 set forth the amino acid sequence for the anti-CD33 VHH DARIC.OX40 fusion protein.

SEQ ID NOs: 52-61 set forth the amino acid sequence for the anti-CD33 VHH DARIC.TNFR2 fusion protein.

SEQ ID NOs: 62-81 set forth the amino acid sequence for the anti-CD33 VHH CAR.

SEQ ID NO:82 shows the amino acid sequence for the anti-CD33 VHH DARIC signaling component.

SEQ ID NO:83 shows a polynucleotide sequence for a Kozak sequence.

SEQ ID NOs: 84-94 show the amino acid sequences of various linkers.

SEQ ID NOs: 95-119 show the amino acid sequences of the protease cleavage site and the self-cleaving polypeptide cleavage site.

In the preceding sequences, when Xaa is present, it may refer to any amino acid or to the absence of an amino acid. In a preferred embodiment, XaaXaa refers to the amino acid sequence SS or KP.

A. Overview

Cancer is one of the leading causes of death worldwide. About 10% of cancers are hematologic cancers, including leukemia, lymphoma, and myeloma. Acute myeloid leukemia (AML) is the most common and fatal blood cancer in adults. Despite major scientific discoveries and new therapies over the past 40 years, treatment outcomes for AML remain negative, especially in the adult patient population. Standard chemotherapy can induce complete remission in selected patients, but the majority of patients eventually relapse and succumb to the disease. In 2012, AML occurred in approximately 351,965 people worldwide and approximately 265,461 died from AML.

CD33 is expressed on most acute myeloid leukemia (AML) cells, leukemia blasts, and possibly leukemia stem cells. CD33 is a difficult target due to its low expression and slow internalization; These properties limit antibody-dependent cell-mediated cytotoxicity and intracellular drug accumulation, which in turn limits the activity of unlabeled antibodies and toxin-carrying antibodies.

The present disclosure generally relates to improved compositions and methods for modulating spatial and temporal modulation of adoptive cell therapy using immunoreceptor complexes (DARICs) that bind to CD33 and modulate with dimerizing agents. Without wishing to be bound by any particular theory, the DARIC compositions and methods contemplated herein offer many advantages over existing CAR T cell therapies in the art, including transduction binding of immune effector cell signals and signaling activity. Including, but not limited to, both spatial and temporal control of DARIC temporal regulation is to prime the DARIC machinery for signal transduction through bridging factor-mediated association of DARIC binding components to DARIC signaling components. DARIC spatial regulation is the binding of the signaling machinery through recognition of CD33 by the DARIC binding domain of the DARIC binding component. In this way, DARIC immune effector cells are activated in the presence of both a target cell expressing CD33 and a bridging factor.

The present disclosure also provides improved anti-CD33 CARs that overcome potential limitations of existing CAR T therapies, including, but not limited to, tonicity signaling or antigen-independent signaling, insufficient expression, and/or sub-therapeutic activity. It's about architecture.

In various embodiments, the present disclosure contemplates an anti-CD33 VHH DARIC or anti-CD33 VHH CAR that produces an anti-cancer response against a cancer such as AML that expresses CD33 (eg, a full-length CD33 and/or CD33 splice variant). .

In certain embodiments, DARIC comprises: a polypeptide comprising a multimerization domain polypeptide or variant thereof, a transmembrane domain, a costimulatory domain, and/or a primary signaling domain (DARIC signaling component); and a polypeptide comprising an anti-CD33 VHH, a multimerization domain polypeptide or variant thereof, a transmembrane domain, and optionally a costimulatory domain (DARIC binding component). When a bridging factor is present, the DARIC binding component and the signal transduction component associate with each other via the bridging factor to form a functionally active DARIC that targets cells expressing CD33.

In certain embodiments, the DARIC binding component and the multimerization domain of the DARIC signaling component are located extracellularly. Extracellular localization of the multimerization domain offers several advantages over intracellular localization, including more efficient localization of the anti-CD33 VHH domain, higher temporal sensitivity to bridging factor regulation, and non-specific bridging factors. lower toxicities due to the ability to use immunosuppressive doses, but are not limited thereto.

polynucleotides encoding DARIC, a DARIC binding component, and a DARIC signal transduction component; DARIC binding components, DARIC protein complexes, DARIC fusion proteins; cells comprising polynucleotides encoding and/or expressing DARIC, a DARIC binding component, and a DARIC signal transduction component; Methods of using them to treat immune disorders are contemplated herein.

Techniques and related techniques and procedures for recombinant (i.e. engineered) DNA, peptide and oligonucleotide synthesis, immunoassays, tissue culture, transformation (eg, electroporation, lipofection), enzymatic reactions, purification, and related techniques and procedures are described throughout this specification. and as described in various general and more specific references in microbiology, molecular biology, biochemistry, molecular genetics, cell biology, virology, and immunology as cited and discussed throughout. See, eg, Sambrook et al., Molecular Cloning: A Laboratory Manual , 3d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; [ Current Protocols in Molecular Biology (John Wiley and Sons, updated July 2008)]; [ Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology , Greene Pub. Associates and Wiley-Interscience]; Glover, DNA Cloning: A Practical Approach , vol. I & II (IRL Press, Oxford Univ. Press USA, 1985)]; [ Current Protocols in Immunology (Edited by: John E. Coligan, Ada M. Kruisbeek, David H. Margulies, Ethan M. Shevach, Warren Strober 2001 John Wiley & Sons, NY, NY)]; [ Real-Time PCR: Current Technology and Applications , Edited by Julie Logan, Kirstin Edwards and Nick Saunders, 2009, Caister Academic Press, Norfolk, UK]; Anand, Techniques for the Analysis of Complex Genomes , (Academic Press, New York, 1992); Guthrie and Fink, Guide to Yeast Genetics and Molecular Biology (Academic Press, New York, 1991); [ Oligonucleotide Synthesis (N. Gait, Ed., 1984)]; [ Nucleic Acid The Hybridization (B. Hames & S. Higgins, Eds., 1985)]; [ Transcription and Translation (B. Hames & S. Higgins, Eds., 1984)]; [ Animal Cell Culture (R. Freshney, Ed., 1986); Perbal, A Practical Guide to Molecular Cloning (1984)]; [ Next-Generation Genome Sequencing (Janitz, 2008 Wiley-VCH)]; [ PCR Protocols (Methods in Molecular Biology) (Park, Ed., 3rd Edition, 2010 Humana Press)]; [ Immobilized Cells And Enzymes (IRL Press, 1986)]; the treatise, [ Methods In Enzymology (Academic Press, Inc., NY)]; [ Gene Transfer Vectors For Mammalian Cells (JH Miller and MP Calos eds., 1987, Cold Spring Harbor Laboratory)]; Harlow and Lane, Antibodies , (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1998); [ Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987)]; [ Handbook Of Experimental Immunology , Volumes I-IV (DM Weir and CC Blackwell, eds., 1986)]; Roitt, Essential Immunology, 6th Edition, (Blackwell Scientific Publications, Oxford, 1988); [ Current Protocols in Immunology (QE Coligan, AM Kruisbeek, DH Margulies, EM Shevach and W. Strober, eds., 1991)]; [ Annual Review of Immunology ] and other journal articles such as Advances in Immunology are also referenced.

B. Definition

Before presenting the present disclosure in more detail, it may be helpful in understanding the present disclosure to provide definitions of certain terms that will be used herein.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the specific embodiments, preferred embodiments of the compositions, methods, and materials are described herein. For the purposes of this disclosure, the following terms are defined below.

The singular expressions (the articles “a”, “an”, and “the”) are used herein to refer to one or two or more (ie, at least one, or one or more) of the grammatical objects expressed in the singular. For example, “an element” means an element or more than one element.

The use of alternatives (eg, “or”) should be understood to mean one, both, or any combination of the alternatives.

The term “and/or” should be understood to mean either or both of the alternatives.

As used herein, the term “about” or “approximately” refers to at most 15% of a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length. , 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of any quantity, level, value, number, frequency, percentage, dimension, size, amount , weight, or length. As used herein, the terms “about” or “approximately” mean ± 15% of a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length. , ± 10%, ± 9%, ± 8%, ± 7%, ± 6%, ± 5%, ± 4%, ± 3%, ± 2%, or ± 1% of a quantity, level, value, number , frequency, percentage, dimension, size, amount, weight, or length.

In one embodiment, a range (eg, 1 to 5, about 1 to 5, about 1 to about 5) refers to each numerical value subsumed in the range. For example, in one non-limiting and merely illustrative embodiment, the range “1 to 5” includes or 1, 2, 3, 4, 5; or 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, or 5.0; or 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4 , 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0.

As used herein, the term “about” or “approximately” refers to 80%, 85%, 90%, compared to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length; 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or refers to the length. In one embodiment, “substantially the same” refers to an effect approximately equal to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length, e.g., a physiological effect refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that produces

Throughout this specification, unless the context requires otherwise, the words "comprise, comprises, and comprising" mean including the recited step or element or group of steps or elements, but any other step. It will be understood that this does not exclude elements or steps or groups of elements. “Consisting of” means including, but not limited to, those following the phrase “consisting of”. Accordingly, the phrase “consisting of” indicates that the listed elements are required or essential, and that the other elements cannot be present. By “consisting essentially of” it is meant including any of the elements listed following the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the present disclosure for the listed elements. do. Thus, the phrase “consisting essentially of” indicates that the listed elements are required or essential, but that no other elements that materially affect the activity or function of the listed elements are present.

Throughout this specification, “one embodiment, an embodiment,” “a particular embodiment, a certain embodiment,” “a related embodiment,” or “additional embodiment ( an additional embodiment or a further embodiment)” or a combination thereof means that a particular feature, structure, or characteristic described in connection with the implementation is included in at least one implementation. Accordingly, not all of the foregoing phrases appearing in various places throughout this specification refer to essentially the same embodiment. Moreover, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments. It will also be understood that positive recitation of a feature in one implementation serves as a basis for excluding that feature in a particular implementation.

“Antigen (Ag)” refers to compounds, compositions, which can stimulate the production of antibodies or T cell responses in animals, including compositions that are injected or absorbed into animals (eg, compositions comprising cancer-specific proteins). , or a substance. Exemplary antigens include, but are not limited to, lipids, carbohydrates, polysaccharides, glycoproteins, peptides, or nucleic acids. Antigens react with products of specific humoral or cellular immunity, including those induced by a heterologous antigen, such as a disclosed antigen.

“Target antigen” or “target antigen of interest” refers to the portion of CD33 to which a binding domain contemplated herein is designed to bind. In certain embodiments, the target antigen is an epitope of the amino acid sequence set forth in SEQ ID NO:1.

“CD33” refers to a cell surface receptor also known as sialic acid-binding immunoglobulin-like lectin 3 (SIGLEC-3) or GP67. The CD33 gene is located on chromosome 19 and produces a glycosylated protein of about 67 kD. CD33 has two Ig-like domains, one V-set domain and one C2-set domain. CD33 mediates cell-cell interactions and plays a role in maintaining immune cells in a resting state. CD33 recognizes and binds to glycans containing alpha-2,3-linked sialic acid, and even more glycans containing alpha-2,6-linked sialic acid. Upon binding of ligands such as Clq or sialylated glycoproteins, two immunoreceptor tyrosine-based inhibitory motifs (ITIMs) located in the CD33 cytoplasmic tail are phosphorylated by Src-like kinases such as LCK. This phosphorylation provides a docking site for recruitment and activation of the protein-tyrosine phosphatases PTPN6/SHP-1 and PTPN11/SHP-2. CD33 also has at least three identified splice variants. The CD33 ^ΔE2 splice variant lacks the amino acid sequence encoded by exon 2 of the human CD33 gene (amino acids 13-139 of full-length CD33; eg , NP_001076087.1, C2). The CD33 ^7a splice variant lacks 54 carboxy-terminal amino acids (eg, NP_001171079.1) due to an early translation termination signal resident in exon 7a. CD33 ^ΔE2/7a lacks the amino acid encoded by exon 2 and the 54 carboxy-terminal amino acids. CD33 is normally expressed on normal B cells and a subset of activated T cells and natural killer cells, but not on hematopoietic stem cells or outside the hematopoietic system. Both full-length CD33 and/or CD33 splice variants are also expressed in acute myeloid leukemia (AML) blasts of most AML patients.

"Antibody" means at least a light or heavy chain that specifically recognizes and binds to an epitope of an antigen, such as a lipid, carbohydrate, polysaccharide, glycoprotein, peptide, or nucleic acid, containing antigenic determinants such as those recognized by immune cells. refers to a binding agent that is a polypeptide comprising an immunoglobulin variable region.

Reference to “VH” or “VH” refers to the variable region of an immunoglobulin heavy chain or antigen-binding fragment thereof.

“Heavy chain antibody” refers to an antibody that contains two V _H domains and lacks a light chain (Riechmann L. et al., J. Immunol. Methods 231:25-38 (1999); WO94/04678; WO94/25591). (U.S. Patent No. 6,005,079). “Camelid antibody” refers to an antibody isolated from a camel, alpaca, or llama that contains two V _H domains and lacks a light chain. “Humanized VHH” or “humanized camelid antibody” refers to a non-human VHH or camelid antibody that has undergone humanization to reduce the potential immunogenicity of the antibody in human recipients.

“V _H H”, “V _H H antibody” or “V _H H domain” as used herein refers to an antibody fragment containing the smallest known antigen binding unit of the variable region of a heavy chain antibody (Koch-Nolte et al. ( FASEB J. , 21: 3490-3498 (2007)).

“Linker” refers to a plurality of amino acid residues between various polypeptide domains added to maintain proper spacing and conformation of the molecule. In certain embodiments, a linker separates one or more VHH domains, hinge domains, multimerization domains, transmembrane domains, costimulatory domains, and/or primary signal transduction domains.

Illustrative examples of linkers suitable for use in certain embodiments contemplated herein include, but are not limited to, the following amino acid sequences: GGG; DGGGS (SEQ ID NO:84); TGEKP (SEQ ID NO: 85) (see, eg, Liu et al., PNAS 5525-5530 (1997)); GGRR (SEQ ID NO:86) (Pomerantz et al., supra 1995); (GGGGS) _n , where n = 1, 2, 3, 4, or 5 (SEQ ID NO: 87) (Kim et al., PNAS 93, 1156-1160 (1996)); EGKSSGSGSESKVD (SEQ ID NO:88) (Chaudhary et al., 1990, Proc. Natl. Acad. Sci. USA 87:1066-1070); KESGSVSSEQLAQFRSLD (SEQ ID NO: 89) (Bird et al., 1988, Science 242:423-426), GGRRGGGS (SEQ ID NO: 90); LRQRDGERP (SEQ ID NO: 91); LRQKDGGGSERP (SEQ ID NO: 92); LRQKD(GGGS) ₂ ERP (SEQ ID NO:93). Alternatively, the flexible linker may use a computer program capable of modeling both the DNA binding site and the peptide itself (Desjarlais & Berg, PNAS 90:2256-2260 (1993)), or [ PNAS 91:11099-11103] (1994)]) can be reasonably designed by the phage display method. In one embodiment, the linker comprises the following amino acid sequence: GSTSGSGKPGSGEGSTKG (SEQ ID NO: 94) (Cooper et al. Blood , 101(4): 1637-1644 (2003)).

“Spacer domain” refers to a polypeptide that separates two domains. In one embodiment, the spacer domain moves the VHH domain away from the effector cell surface to allow for proper cell/cell contact, antigen binding, and activation (Patel et al., Gene Therapy , 1999; 6: 412-419). ] Reference). In certain embodiments, a spacer domain separates one or more VHH domains, multimerization domains, transmembrane domains, costimulatory domains, and/or primary signal transduction domains. Spacer domains may be derived from natural, synthetic, semisynthetic, or recombinant sources. In certain embodiments, the spacer domain is part of an immunoglobulin including, but not limited to, one or more heavy chain constant regions (eg, CH2 and CH3). The spacer domain may comprise the amino acid sequence of a naturally occurring immunoglobulin hinge region or an altered immunoglobulin hinge region.

“Hinge domain” refers to a polypeptide that is responsible for positioning the antigen binding domain away from the effector cell surface to allow for proper cell/cell contact, antigen binding, and activation. In certain embodiments, a polypeptide may comprise one or more hinge domains between a binding domain and a multimerization domain, between a binding domain and a transmembrane domain (TM), or between a multimerization domain and a transmembrane domain. The hinge domain may be derived from a natural, synthetic, semisynthetic, or recombinant source. The hinge domain may comprise the amino acid sequence of a naturally occurring immunoglobulin hinge region or an altered immunoglobulin hinge region.

“Multimerization domain” as used herein refers to a polypeptide that preferentially interacts or binds with another different polypeptide either directly or through a crosslinking molecule (eg, a chemically inducible dimerizing agent), wherein the different multimerization domains The interaction of effectively promote Multimerization domains can be derived from natural, synthetic, semisynthetic, or recombinant sources.

Illustrative examples of suitable multimerization domains for use in the specific embodiments contemplated herein are FK506 binding protein (FKBP) polypeptides or variants thereof, FKBP-rapamycin binding (FRB) polypeptides or variants thereof, calcineurin polypeptides or variants thereof variant, cyclophilin polypeptide or variant thereof, bacterial dihydrofolate reductase (DHFR) polypeptide or variant thereof, PYR1-like 1 (PYL1) polypeptide or variant thereof, abscisic acid insensitive 1 (ABI1) polypeptide or variant thereof, GIB1 polypeptide or variants thereof, or GAI polypeptides or variants thereof.

As used herein, the term “FKBP-rapamycin binding polypeptide” refers to an FRB polypeptide. In certain embodiments, the FRB polypeptide is a FKBP12-rapamycin binding polypeptide. FRB polypeptides suitable for use in certain embodiments contemplated herein generally contain at least about 85 to about 100 amino acid residues. In certain embodiments, the FRB polypeptide comprises 93 amino acids from Ile-2021 to Lys-2113 with respect to GenBank Accession No. L34075.1 and a mutation in T2098L. The FRB polypeptides contemplated herein bind to the FKBP polypeptide via a bridging factor to form a ternary complex.

As used herein, the term “FK506 binding protein” refers to a FKBP polypeptide. In certain embodiments, the FKBP polypeptide is a FKBP12 polypeptide or a FKBP12 polypeptide comprising a F36V mutation. In certain embodiments, the FKBP domain may also be referred to as a “rapamycin binding domain”. Information regarding the nucleotide sequences, cloning, and other aspects of various FKBP species is known in the art (eg, Staendart et al., Nature 346:671, 1990 (human FKBP12); Kay, Biochem. J. 314:361, 1996]). The FKBP polypeptides contemplated herein bind to the FRB polypeptide via a bridging factor to form a ternary complex.

A “bridging factor” refers to a molecule that binds to and is placed between two or more multimerization domains. In certain embodiments, the multimerization domain substantially contributes to or efficiently promotes the formation of a polypeptide complex only in the presence of a crosslinking factor. In certain embodiments, the multimerization domain does not contribute to or efficiently promote the formation of a polypeptide complex in the absence of a crosslinking factor. Illustrative examples of suitable crosslinking factors for use in certain embodiments contemplated herein include APAP21967, rapamycin (sirolimus) or a rapalog thereof, cumermycin or a derivative thereof, gibberellin or a derivative thereof, abscisic acid (ABA) ) or a derivative thereof, methotrexate or a derivative thereof, cyclosporin A or a derivative thereof, FKCsA or a derivative thereof, a trimethoprim (Tmp)-synthetic ligand for FKBP (SLF) or a derivative thereof, or any combination thereof; It is not limited to these.

Rapamycin analogues (rapalogs) include, but are not limited to, those described in US Pat. No. 6,649,595, these rapalog structures are incorporated herein by reference in their entirety. In certain embodiments, the bridging factor is a rapalog with substantially reduced immunosuppressive effect compared to rapamycin. In a preferred embodiment, the rapalog is AP21967 (also known as C-16-(S)-7-methylindolapamycin, a chemically modified non-immunosuppressive rapamycin analogue, IC50=10 nM). Other exemplary rapalogs suitable for use in certain embodiments contemplated herein are everolimus, novolimus, pimecrolimus, ridaforolimus, tacrolimus (tacrolimus), temsirolimus, umirolimus, and zotarolimus.

A "substantially reduced immunosuppressive effect", as measured clinically or in an appropriate in vitro (eg, inhibition of T cell proliferation) or in vivo surrogate of human immunosuppressive activity, is at least 0.1 for the same dose. to 0.005 fold lower immunosuppressive effect observed or expected.

A “transmembrane domain” or “TM domain” is a domain that anchors a polypeptide to the plasma membrane of a cell. TM domains can be derived from natural, synthetic, semisynthetic, or recombinant sources.

The term “effector function” or “effector cell function” refers to the specialized function of an immune effector cell. Effector functions include, but are not limited to, activation, cytokine production, proliferation and cytotoxic activity, and include, but are not limited to, release of cytotoxic factors, or other cellular responses induced by antigen binding to receptors expressed in immune effector cells.

An “intracellular signal transduction domain” or “endodomain” refers to a portion of a protein that transduces effector function signals and directs a cell to perform a specific function. In general, the entire intracellular signaling domain can be used, but in many cases it is not necessary to use the entire domain. As to the extent to which a truncated portion of an intracellular signal transduction domain is used, it may be used instead of the entire domain as long as such truncated portion transduces an effector function signal. The term “intracellular signaling domain” is meant to include any truncated portion of an intracellular signaling domain necessary or sufficient to transduce an effector function signal.

Signals generated only through TCRs are insufficient for full activation of T cells, and it is known that secondary or costimulatory signals are also required. Thus, it can be said that T cell activation is mediated by two distinct classes of intracellular signaling domains: primary signaling domains that initiate antigen-dependent primary activation via TCRs (eg, the TCR/CD3 complex); and a co-stimulatory signaling domain that acts in an antigen-independent manner to provide a secondary or co-stimulatory signal.

“Primary signaling domain” refers to an intracellular signaling domain that regulates primary activation of the TCR complex in a stimulatory or inhibitory manner. A primary signaling domain that acts in a stimulatory manner may comprise a signaling motif known as an immunoreceptor tyrosine-based activation motif or ITAM. Illustrative examples of ITAMs containing a primary signaling domain suitable for use in certain embodiments include, but are not limited to, those derived from FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d. not limited

As used herein, the term “costimulatory signaling domain” or “costimulatory domain” refers to the intracellular signaling domain of a costimulatory molecule. Costimulatory molecules are cell surface molecules other than antigen receptors or Fc receptors that, when bound to antigen, provide a second signal necessary for efficient activation and function of T lymphocytes. Illustrative examples of such costimulatory molecules from which the costimulatory domain may be isolated include, but are not limited to: Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7 , TLR8, TLR9, TLR10, caspase recruitment domain family member 11 (CARD11), CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD94, CD134 (OX40), CD137 (4-1BB) , CD278 (ICOS), DNAX-activating protein 10 (DAP10), T cell lineage member 1 (LAT), leukocyte protein of 76 kD containing SH2 domain (SLP76), T cell receptor-associated transmembrane adapter 1 (TRAT1), TNFR2, TNF receptor superfamily member 14 (TNFRS14; HVEM), TNF receptor superfamily member 18 (TNFRS18; GITR), TNF receptor superfamily member 25 (TNFRS25; DR3), and the zeta chain of T cell receptor associated protein kinase 70 (ZAP70) .

An “immune disorder” refers to a disease that elicits a response from the immune system. In certain embodiments, the term “immune disorder” refers to cancer, autoimmune disease, or immunodeficiency.

As used herein, the term “cancer” generally relates to a class of diseases or conditions in which abnormal cells can divide uncontrolled and invade adjacent tissues.

As used herein, the term “malignant” refers to uncontrolled growth (ie, division beyond normal limits), infiltration (ie, invasion and destruction of adjacent tissue), and metastasis (ie, lymph or blood) of a tumor cell population. cancer that has spread to other locations in the body through As used herein, the term “metastasis” refers to the spread of cancer from one part of the body to another. Tumors formed by diffused cells are referred to as “metastatic tumors” or “metastasis”. Metastatic tumors contain cells similar to those in the original (primary) tumor.

As used herein, the terms “benign” or “non-malignant” refer to tumors that can grow larger but do not spread to other parts of the body. Benign tumors are self-limiting and usually do not invade or metastasize.

A “cancer cell” refers to an individual cell of a cancerous growth or tissue. Cancer cells include both solid and liquid cancers. A “tumor” or “tumor cell” refers to a swelling or lesion formed by abnormal growth of cells, which may generally be benign, premalignant, or malignant. Most cancers form tumors, but liquid cancers such as leukemia do not necessarily form tumors. For cancers that form tumors, the terms cancer (cell) and tumor (cell) are used interchangeably. The amount of a tumor in an individual is the “tumor burden,” which can be measured as the number, volume, or weight of tumors.

The term “relapse” refers to the return of cancer after a period of improvement or remission, or diagnosis of the signs and symptoms of return.

"Remission" is also referred to as "clinical remission" and includes both partial and complete remission. In partial remission, some, but not all, signs and symptoms of the cancer have disappeared. In complete remission, the cancer may still be in the body, but all signs and symptoms of the cancer are gone.

“Refractory” refers to cancer that is resistant or does not respond to therapy with a particular therapeutic agent. A cancer may be refractory from the initiation of treatment (ie, non-responsive from initial exposure to the therapeutic agent), or may become refractory by developing resistance to the therapeutic agent over the first treatment period or during subsequent treatment periods.

As used herein, the terms "individual" and "subject" are often used interchangeably, and any animal exhibiting symptoms of cancer or other immune disorder that can be treated with the compositions and methods contemplated elsewhere herein. refers to Suitable subjects (eg, patients) include laboratory animals (eg, mice, rats, rabbits, or guinea pigs), farm animals, and livestock or pets (eg, cats or dogs). Non-human primates and preferably human patients are included. Typical subjects include human patients suffering from, diagnosed with, or at risk of developing cancer or other immune disorders.

As used herein, the term “patient” refers to a subject diagnosed with cancer or other immune disorder that can be treated with the compositions and methods disclosed elsewhere herein.

As used herein, “treatment or treating” includes any beneficial or desirable effect on the symptoms or pathology of a disease or pathological condition, and includes one or more measurable markers of the disease or condition being treated. It can contain very minimal reductions. Treatment may optionally include reducing the disease or condition, or delaying the progression of the disease or condition, eg, delaying tumor proliferation. “Treatment” does not necessarily refer to complete eradication or cure of the disease or condition, or symptoms associated therewith.

As used herein, “prevention (like words such as prevent or prevented or preventing, etc.)” refers to an approach for preventing, suppressing, or reducing the likelihood of the onset or recurrence of a disease or condition. It also refers to delaying the onset or recurrence of a disease or condition or delaying the onset or recurrence of symptoms of a disease or condition. As used herein, “prevention” and like words also include reducing the intensity, effect, symptoms, and/or burden of a disease or condition prior to the onset or recurrence of the disease or condition.

As used herein, the phrase “alleviating at least one symptom” refers to reducing one or more symptoms of a disease or condition for which a subject is being treated. In certain embodiments, the disease or condition being treated is cancer, wherein one or more symptoms to be alleviated are weakness, fatigue, shortness of breath, easily bruising and bleeding, frequent infections, enlarged lymph nodes, bloating (due to an enlarged abdominal organ), or abdominal pain, bone or joint pain, fractures, unintentional weight loss, loss of appetite, night sweats, persistent low fever, and decreased urination (due to impaired renal function).

The terms “enhance” or “promote” or “increase” or “expand” refer to a greater physiological response (i.e., downstream refers generally to the ability of a composition contemplated herein to produce, induce, or cause an effect). A measurable physiological response may include an increase in the ability of T cell proliferation, activation, persistence, cytokine secretion, and/or cancer cell killing, among other things apparent from understanding in the art and the description herein. An “increased” or “enhanced” amount is typically an amount that is “statistically significant” and represents 1.1, 1.2, 1.5, 2, 3 of the response produced by the vehicle or control composition. , 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more (e.g., by 500, 1000) increments (any integer greater than 1) values and decimal values between integer values, including 1.5, 1.6, 1.7, 1.8, etc.).

The term "decrease or lessen or reduce or abate" is used herein to produce, induce, or elicit less physiological response (i.e., a downstream effect) compared to the response elicited by the vehicle or control molecule/composition. Refers generally to the ability of the composition under consideration. A "decreased or reduced" amount is typically a "statistically significant" amount and is equivalent to 1.1, 1.2, 1.5, 2, the response produced by the vehicle or control composition (baseline response) or the response in a particular cell lineage. 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more (e.g., by 500, 1000) reduced (all greater than 1) integer values and decimal values between integer values, including 1.5, 1.6, 1.7, 1.8, etc.).

"maintain or maintenance" or "preserve" or "no change" or "no substantial change" or "no substantial decrease" means vehicle or control Refers to the ability of a composition contemplated herein to produce, induce, or elicit a substantially similar or similar physiological response (ie, a downstream effect) compared to the response caused by the molecule/composition. A comparable response is one that differs significantly from the baseline response or has no measurable difference.

Additional definitions are set forth throughout this disclosure.

C. CD33 VHH DARIC

In certain embodiments, a DARIC receptor comprising an anti-CD33 VHH domain that redirects cytotoxicity of immune effector cells towards cancer cells expressing CD33 is contemplated. As used herein, the terms “CD33 VHH DARIC receptor”, “anti-CD33 VHH DARIC receptor”, “CD33 VHH DARIC”, or “anti-CD33 VHH DARIC” are used interchangeably and refer to full-length CD33 or CD33 Target cells expressing splice variants and transducing immunostimulatory signals in immune effector cells upon exposure to multimerizing agents or bridging factors (e.g., stimulating immune effector cell activity and function, and production of proinflammatory cytokines) and/or increase secretion). In a preferred embodiment, the CD33 VHH DARIC is a multi-chain chimeric receptor comprising a DARIC binding component and a DARIC signaling component comprising a VHH domain that recognizes full-length CD33 and/or CD33 splice variants.

In one embodiment, the DARIC signaling component and the DARIC binding component are expressed from the same cell. In another embodiment, the DARIC signaling component and the DARIC binding component are expressed from different cells. In certain embodiments, the DARIC signaling component is expressed from a cell and the DARIC binding component is supplied exogenously as a polypeptide. In one embodiment, a DARIC binding component preloaded with a bridging factor is supplied exogenously to a cell expressing a DARIC signaling component.

1. CD33 DARIC Signaling Component

The terms "DARIC signal transduction component", "CD33 DARIC signal transduction component", "DARIC signal transduction polypeptide", "or "DARIC signal transduction polypeptide" are used interchangeably and include one or more multimerization domains, a transmembrane domain, and one or more intracellular signal transduction domains.In certain embodiments, the DARIC signal transduction component comprises a multimerization domain, a transmembrane domain, a costimulatory domain, and/or a primary signal transduction domain. In an embodiment, the DARIC signaling component comprises a first multimerization domain, a first transmembrane domain, a first costimulatory domain, and/or a primary signaling domain.

In certain embodiments, the DARIC signal transduction component comprises one or more multimerization domains.

Illustrative examples of multimerization domains suitable for use in certain CD33 DARIC signaling components contemplated herein include FK506 binding protein (FKBP) polypeptides or variants thereof, FKBP-rapamycin binding (FRB) polypeptides or variants thereof, calcineurin polypeptide or variant thereof, cyclophilin polypeptide or variant thereof, bacterial dihydrofolate reductase (DHFR) polypeptide or variant thereof, PYR1-like 1 (PYL1) polypeptide or variant thereof, abscisic acid insensitive 1 (ABI1) polypeptide or variant thereof including, but not limited to.

In certain embodiments, the CD33 DARIC signaling component comprises a FRB polypeptide.

In certain preferred embodiments, the CD33 DARIC signaling component comprises an FRB polypeptide comprising the T2098L mutation or a variant thereof. In certain preferred embodiments, the CD33 DARIC signaling component comprises a FKBP12 polypeptide or variant thereof.

In some embodiments, the CD33 VHH DARIC signaling component comprises a hinge domain.

Exemplary hinge domains suitable for use in the CD33 VHH DARIC signaling component described herein include hinge regions derived from the extracellular regions of type 1 membrane proteins such as CD28, CD8α, and CD4, wherein the hinge regions are It may be the wild-type hinge region of the molecule or it may be altered.

In certain embodiments, the DARIC signaling component comprises a transmembrane domain.

In certain embodiments, the DARIC signaling component comprises a hinge domain and a transmembrane domain.

Illustrative examples of transmembrane domains suitable for use in certain CD33 DARIC signaling components contemplated herein include the alpha, beta, gamma, or delta chains of T cell receptors, CD3ε, CD3ζ, CD4, CD5, CD8α, CD9, CD 16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD71, CD80, CD86, CD 134, CD137, CD152, CD 154, amnionless (AMN), and programmed cell death 1 (PDCD1) including but not limited to the transmembrane domain(s) of In a preferred embodiment, the CD33 DARIC signaling component comprises a CD4 transmembrane domain. In a preferred embodiment, the CD33 DARIC signaling component comprises a CD8α transmembrane domain.

In certain embodiments, the DARIC signaling component comprises a linker connecting the C-terminus of the transmembrane domain to the N-terminus of the intracellular signaling domain. In various preferred embodiments, a short oligopeptide linker or polypeptide linker, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in length, comprises a transmembrane domain and an intracellular signal transduction domain. connect the Glycine-serine based linkers provide particularly suitable linkers.

A DARIC signaling component contemplated in certain embodiments herein comprises one or more intracellular signaling domains. In one embodiment, the CD33 DARIC signaling component comprises one or more costimulatory signaling domains and/or primary signaling domains. In one embodiment, the intracellular signaling domain comprises an immunoreceptor tyrosine activation motif (ITAM).

Illustrative examples of ITAMs containing primary signaling domains suitable for use in certain CD33 DARIC signaling components contemplated herein are from FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d. derived from, but not limited to. In a preferred embodiment, the CD33 DARIC signaling component comprises a CD3ζ primary signaling domain and one or more costimulatory signaling domains. The primary signaling domain and the costimulatory signaling domain may be serially linked to the carboxyl terminus of the transmembrane domain in any order.

Illustrative examples of suitable costimulatory domains for use in certain CD33 DARIC signaling components contemplated herein include, but are not limited to, domains isolated from the following costimulatory molecules: Toll-like receptor 1 (TLR1) ), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, caspase recruitment domain family member 11 (CARD11), CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD94, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DNAX-activating protein 10 (DAP10), T cell lineage member 1 (LAT), leukocyte protein of 76 kD (SLP76) containing SH2 domain, T Cell receptor associated transmembrane adapter 1 (TRAT1), TNFR2, TNF receptor superfamily member 14 (TNFRS14; HVEM), TNF receptor superfamily member 18 (TNFRS18; GITR), TNF receptor superfamily member 25 (TNFRS25; DR3), and Zeta chain of T cell receptor associated protein kinase 70 (ZAP70).

In certain embodiments, a CD33 DARIC signal transduction component contemplated herein comprises a signal peptide. Illustrative examples of suitable signal peptides for use in certain CD33 DARIC signaling components include, but are not limited to, an IgG1 heavy chain signal polypeptide, an Igκ light chain signal polypeptide, a CD8α signal polypeptide, or a human GM-CSF receptor alpha signal polypeptide. does not In various preferred embodiments, the CD33 DARIC signal transduction component comprises a CD8α signal polypeptide.

In certain embodiments, the CD33 DARIC signaling component comprises one or more costimulatory signaling domains selected from the group consisting of CD28, CD137, and CD134. In certain embodiments, the CD33 DARIC signaling component comprises one or more costimulatory signaling domains selected from the group consisting of CD28, CD137, and CD134, and a CD3ζ primary signaling domain. In certain embodiments, the CD33 DARIC signaling component comprises a CD137 costimulatory domain and a CD3ζ primary signaling domain.

In certain embodiments, the CD33 DARIC signaling component comprises a FRB T2098L multimerization domain, a CD8α transmembrane domain, a CD137 costimulatory domain, and a CD3ζ primary signaling domain.

In a preferred embodiment, the CD33 VHH DARIC signaling component comprises the amino acid sequence set forth in SEQ ID NO:82.

2. CD33 DARIC binding component

"DARIC binding component", "DARIC binding polypeptide", "CD33 VHH DARIC binding component", or "CD33 VHH DARIC binding polypeptide" are used interchangeably, and include an anti-CD33 VHH domain and one or more multimerizing domains. refers to a polypeptide. In certain embodiments, the CD33 VHH DARIC binding component comprises an anti-CD33 VHH domain, a multimerization domain, and a transmembrane domain. In certain embodiments, the CD33 VHH DARIC binding component comprises an anti-CD33 VHH domain, a second multimerization domain, and a second transmembrane domain. In certain other embodiments, the CD33 VHH DARIC binding component comprises an anti-CD33 VHH domain, a multimerization domain, a transmembrane domain, and one or more intracellular signaling domains. In certain embodiments, the CD33 VHH DARIC binding component comprises an anti-CD33 VHH domain, a second multimerization domain, a second transmembrane domain, and a second costimulatory domain.

In certain embodiments, the CD33 VHH DARIC binding component comprises one or more anti-CD33 VHH domains.

In a particularly preferred embodiment, the anti-CD33 VHH domain is a humanized camelid VHH. In certain embodiments, the anti-CD33 VHH domain is a humanized camelid VHH that binds to one or more epitopes of full-length CD33 (SEQ ID NO: 1) or one or more epitopes of a CD33 splice variant. In certain embodiments, the anti-CD33 VHH domain is a humanized camelid VHH that binds to the same one or more epitopes present in both full-length CD33 and CD33 splice variants.

In certain preferred embodiments, the anti-CD33 VHH domain is a humanized camelid VHH comprising the amino acid sequence set forth in any one of SEQ ID NOs: 3-6, 10-11, 13-16, and 20-21. In certain preferred embodiments, the anti-CD33 VHH domain is a humanized camelid VHH comprising the amino acid sequence set forth in SEQ ID NO:10. In certain preferred embodiments, the anti-CD33 VHH domain is a humanized camelid VHH comprising the amino acid sequence set forth in SEQ ID NO:20.

In certain embodiments, the DARIC binding component comprises one or more multimerization domains.

Illustrative examples of multimerization domains suitable for use in certain CD33 VHH DARIC binding components contemplated herein include FKBP polypeptides or variants thereof, FRB polypeptides or variants thereof, calcineurin polypeptides or variants thereof, cyclophilin polypeptides or variants thereof, DHFR polypeptide or variant thereof, PYL1 polypeptide or variant thereof, ABI1 polypeptide or variant thereof.

In certain embodiments, the CD33 VHH DARIC binding component comprises a FRB polypeptide or variant thereof, and the DARIC signaling component comprises a FKBP polypeptide or variant thereof. In a preferred embodiment, the CD33 VHH DARIC binding component comprises a FRB polypeptide comprising the T2098L mutation or a variant thereof, and the DARIC signaling component comprises a FKBP12 polypeptide or a variant thereof.

In certain embodiments, the CD33 VHH DARIC binding component comprises a FKBP polypeptide or variant thereof, and the DARIC signaling component comprises a FRB polypeptide or variant thereof. In a preferred embodiment, the CD33 VHH DARIC binding component comprises a FKBP12 polypeptide or variant thereof, and the DARIC signaling component comprises a FRB polypeptide comprising the T2098L mutation or a variant thereof.

In some embodiments, the CD33 VHH DARIC binding component comprises a hinge domain.

Exemplary hinge domains suitable for use in the CD33 VHH DARIC binding components described herein include hinge regions derived from the extracellular regions of type 1 membrane proteins such as CD28, CD8α, and CD4, wherein the hinge region comprises these molecules It may be a wild-type hinge region of , or it may be altered.

In certain embodiments, the DARIC binding component comprises a transmembrane domain. In certain embodiments, the DARIC binding component comprises a hinge domain and a transmembrane domain. In one embodiment, the transmembrane domain may be the same as the transmembrane domain used in the DARIC signaling component. In one embodiment, the transmembrane domain may be different from the transmembrane domain used in the DARIC signal transduction component.

Illustrative examples of transmembrane domains suitable for use in certain CD33 VHH DARIC binding components contemplated herein include alpha, beta, gamma, or delta chains of T cell receptors, CD3ε, CD3ζ, CD4, CD5, CD8α, CD9, CD 16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD71, CD80, CD86, CD 134, CD137, CD152, CD 154, amnionless (AMN), and programmed cell death 1 (PDCD1) including but not limited to the transmembrane domain(s) of In a preferred embodiment, the CD33 DARIC binding component comprises a CD8α transmembrane domain. In a preferred embodiment, the CD33 VHH DARIC binding component comprises a CD4 transmembrane domain.

In various preferred embodiments, a short oligopeptide linker or polypeptide linker, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in length, comprises a transmembrane domain and an intracellular signal transduction domain. connect the Glycine-serine based linkers provide particularly suitable linkers.

A DARIC binding component contemplated in certain embodiments herein does not comprise one or more intracellular signal transduction domains.

In certain other embodiments, a CD33 VHH DARIC binding component contemplated herein comprises one or more intracellular signaling domains. In preferred embodiments wherein the CD33 VHH DARIC binding component comprises one or more intracellular signaling domains, these domains are different from the intracellular signaling domain present in the cognate CD33 DARIC signaling component. In one embodiment, the CD33 VHH DARIC binding component comprises a costimulatory signaling domain.

Illustrative examples of suitable costimulatory domains for use in certain CD33 VHH DARIC binding components contemplated herein include, but are not limited to, domains isolated from the following costimulatory molecules: Toll-like receptor 1 (TLR1) ), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, caspase recruitment domain family member 11 (CARD11), CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD94, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DNAX-activating protein 10 (DAP10), T cell lineage member 1 (LAT), leukocyte protein of 76 kD (SLP76) containing SH2 domain, T Cell receptor associated transmembrane adapter 1 (TRAT1), TNFR2, TNF receptor superfamily member 14 (TNFRS14; HVEM), TNF receptor superfamily member 18 (TNFRS18; GITR), TNF receptor superfamily member 25 (TNFRS25; DR3), and Zeta chain of T cell receptor associated protein kinase 70 (ZAP70). In a preferred embodiment, the costimulatory domain is derived from, obtained or isolated from TNFR2 or OX40.

In certain embodiments, a DARIC binding component contemplated herein comprises a signal peptide. Illustrative examples of signal peptides suitable for use in certain CD33 VHH DARIC binding components include, but are not limited to, an IgG1 heavy chain signal polypeptide, an Igκ light chain signal polypeptide, a CD8α signal polypeptide, or a human GM-CSF receptor alpha signal polypeptide. does not In various preferred embodiments, the CD33 VHH DARIC binding component comprises a CD8α signal polypeptide.

In certain embodiments, the CD33 VHH DARIC binding component comprises a VHH domain that binds CD33, a FKBP12 multimerization domain, and a CD4 transmembrane domain, and optionally a costimulatory domain.

In certain embodiments, the CD33 VHH DARIC binding component comprises a VHH that binds CD33, and a FKBP12 multimerization domain.

In some embodiments, the CD33 VHH DARIC binding component comprises a VHH domain comprising the amino acid sequence set forth in any one of SEQ ID NOs: 2-21, a FKBP12 multimerization domain, and a CD4 transmembrane domain, and optionally a costimulatory domain.

In some embodiments, the CD33 VHH DARIC binding component comprises a VHH domain comprising the amino acid sequence set forth in SEQ ID NO:10 or SEQ ID NO:20, a FKBP12 multimerization domain, and a CD4 transmembrane domain, and optionally a costimulatory domain.

In some embodiments, the CD33 VHH DARIC binding component comprises a VHH domain comprising the amino acid sequence set forth in any one of SEQ ID NOs: 2-21, and a FKBP12 multimerization domain.

In some embodiments, the CD33 VHH DARIC binding component comprises a VHH domain comprising the amino acid sequence set forth in SEQ ID NO: 10 or 20, and a FKBP12 multimerization domain.

In some embodiments, the CD33 VHH DARIC binding component comprises an amino acid sequence set forth in any one of SEQ ID NOs: 22-31.

In some embodiments, the CD33 VHH DARIC binding component comprises the amino acid sequence set forth in SEQ ID NO:30.

3. Crosslinking factor

A bridging factor contemplated in certain embodiments herein mediates or promotes association between the CD33 DARIC signal transduction component and the CD33 VHH DARIC binding component through the multimerization domain of each of these components. A bridging factor binds to and is disposed between the multimerization domains to promote binding of the CD33 DARIC signaling component to the CD33 VHH DARIC associative component. When the bridging factor is present, the CD33 VHH DARIC binding component and the CD33 DARIC signaling component associate and initiate immune effector cell activity against the target cell when the CD33 VHH DARIC binding component binds to CD33 expressed on the target cell. In the absence of a bridging factor, the CD33 VHH DARIC binding component does not associate with the CD33 DARIC signaling component and CD33 VHH DARIC is not activated.

In certain embodiments, the CD33 DARIC signaling component and the CD33 VHH DARIC binding component comprise a cognate pair of multimerization domains selected from the group consisting of: FKBP and FKBP12-rapamycin binding (FRB), FKBP and calcineurin, FKBP and cyclophilin, FKBP and bacterial dihydrofolate reductase (DHFR), calcineurin and cyclophilin, PYR1-like 1 (PYL1) and abscisic acid insensitive 1 (ABI1).

In certain embodiments, the multimerization domain of the CD33 VHH DARIC signaling component and the binding component binds to a crosslinking factor selected from the group consisting of: rapamycin or a rapalog thereof, cumermycin or a derivative thereof, gibberellin or a derivative thereof, Abcisic acid (ABA) or derivatives thereof, methotrexate or derivatives thereof, cyclosporine A or derivatives thereof, FK506/cyclosporine A (FKCsA) or derivatives thereof, and trimethoprim (Tmp)-synthesis for FK506 binding protein (FKBP) Ligand (SLF) or a derivative thereof.

In certain embodiments, the CD33 DARIC signaling component and the CD33 VHH DARIC binding component comprise one or more FRB and/or FKBP multimerization domains or variants thereof. In certain embodiments, the CD33 DARIC signaling component comprises a FRB multimerization domain or variant thereof, and the CD33 VHH DARIC binding component comprises a FKBP multimerization domain or variant thereof. In certain embodiments, the CD33 DARIC signaling component comprises a FRB T2098L multimerization domain or variant thereof, and the CD33 VHH DARIC binding component comprises a FKBP12 or FKBP12 F36V multimerization domain or a variant thereof.

Illustrative examples of suitable crosslinking factors for use in certain embodiments contemplated herein are AP1903, AP20187, AP21967 (also known as C-16-(S)-7-methylindolapamycin), everolimus, novol Remus, pimecrolimus, ridaforolimus, tacrolimus, temsirolimus, umirolimus and zotarolimus. In a particularly preferred embodiment, the crosslinking factor is AP21967. In certain preferred embodiments, the bridging factor is a non-immunosuppressive dose of sirolimus (rapamycin).

D. Anti-CD33 Chimeric Antigen Receptor

In certain embodiments, immune effector cells contemplated herein comprise an anti-CD33 VHH CAR. Chimeric antigen receptors (CARs) are molecules that combine antibody-based specificity for a target antigen (eg, a tumor antigen) with a T cell receptor-activating intracellular domain to generate a chimeric protein that exhibits specific anti-tumor cell immune activity. As used herein, the term “chimeric” describes being composed of different proteins or portions of DNA from different origins.

In certain embodiments, the T cell is engineered by introducing a polynucleotide encoding an anti-CD33 VHH CAR.

In certain embodiments, the T cell is engineered by introducing a vector comprising a polynucleotide encoding an anti-CD33 VHH CAR.

In various embodiments, the anti-CD33 CAR comprises a VHH domain that binds CD33, a transmembrane domain, and one or more intracellular signaling domains. A key property of CARs is by reinducing immune effector cell specificity, resulting in proliferation, cytokine production, phagocytosis or production of molecules capable of mediating apoptosis of target antigen-expressing cells in a manner independent of major histocompatibility (MHC). and their ability to exploit the cell-specific targeting capabilities of monoclonal antibodies, soluble ligands, or cell-specific co-receptors.

In some embodiments, the anti-CD33 VHH CAR comprises a spacer domain. In certain embodiments, the spacer domain comprises CH2 and CH3 of an IgG1, IgG4, or IgD.

Exemplary hinge domains suitable for use in the anti-CD33 VHH CARs described herein include hinge regions derived from the extracellular regions of type 1 membrane proteins such as CD28, CD8α, and CD4, wherein the hinge region comprises these molecules. It may be a wild-type hinge region of , or it may be altered. In another embodiment, the hinge domain comprises a CD8α hinge region.

The transmembrane (TM) domain of the CAR fuses the extracellular binding moiety with the intracellular signaling domain and anchors the CAR to the plasma membrane of immune effector cells. TM domains can be derived from natural, synthetic, semisynthetic, or recombinant sources.

Exemplary TM domains include the alpha, beta, gamma, or delta chain of a T cell receptor, CD3ε, CD3ζ, CD4, CD5, CD8α, CD9, CD 16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD71, CD80, CD86, CD 134, CD137, CD152, CD 154, AMN, and PDCD1 (ie, may comprise at least transmembrane region(s) thereof).

In one embodiment, the anti-CD33 VHH CAR comprises a TM domain derived from CD8α. In another embodiment, the CAR contemplated herein is a TM domain derived from CD8α, and preferably 1, 2, 3, 4, 5, 6, 7, linking the TM domain and the intracellular signaling domain of the CAR, oligo-linkers or polypeptide linkers of 8, 9, or 10 amino acids in length. Glycine-serine linkers provide particularly suitable linkers.

In a preferred embodiment, the anti-CD33 VHH CAR comprises an intracellular signaling domain comprising one or more costimulatory signaling domains and a primary signaling domain.

A primary signaling domain that acts in a stimulatory manner may comprise a signaling motif known as an immunoreceptor tyrosine-based activation motif or ITAM.

Exemplary embodiments of ITAMs containing primary signaling domains suitable for use in anti-CD33 VHH CARs contemplated in certain embodiments include FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d include those derived from In certain preferred embodiments, the CAR comprises a CD3ζ primary signaling domain and one or more costimulatory signaling domains. The intracellular primary signaling domain and the costimulatory signaling domain may be tandemly linked to the carboxyl terminus of the transmembrane domain in any order.

In certain embodiments, the anti-CD33 VHH CAR comprises one or more costimulatory signaling domains for enhancing the efficacy and proliferation of T cells expressing the CAR receptor.

Illustrative examples of such costimulatory molecules suitable for use in anti-CD33 VHH CARs contemplated in certain embodiments include TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD94, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DAP10, LAT, SLP76, TRAT1, TNFR2, and ZAP70 However, it is not limited to these. In one embodiment, the CAR comprises one or more costimulatory signaling domains selected from the group consisting of CD28, CD137, and CD134, and a CD3ζ primary signaling domain.

In various embodiments, the anti-CD33 VHH CAR comprises: a VHH that binds CD33; A transmembrane domain isolated from a polypeptide selected from the group consisting of: CD4, CD8α, CD154, and PD-1; one or more intracellular costimulatory signaling domains isolated from a polypeptide selected from the group consisting of: CD28, CD134, and CD137; and a signal transduction domain isolated from a polypeptide selected from the group consisting of: FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d.

In various embodiments, the anti-CD33 VHH CAR comprises: a VHH that binds CD33; A transmembrane domain isolated from a polypeptide selected from the group consisting of: CD4, CD8α, CD154, and PD-1; one or more intracellular costimulatory signaling domains isolated from a polypeptide selected from the group consisting of: CD28, CD134, and CD137; and a signal transduction domain isolated from a polypeptide selected from the group consisting of: FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d.

In a preferred embodiment, the anti-CD33 VHH CAR comprises a VHH comprising the amino acid sequence set forth in any one of SEQ ID NOs: 2-21, a CD8α hinge domain, a CD8α transmembrane domain, a 4-1BB costimulatory domain, and a CD3ζ primary signaling domain. includes

In a preferred embodiment, the anti-CD33 VHH CAR comprises a VHH comprising the amino acid sequence set forth in SEQ ID NO: 10, a CD8α hinge domain, a CD8α transmembrane domain, a 4-1BB costimulatory domain, and a CD3ζ primary signaling domain.

In certain embodiments, the anti-CD33 VHH CAR comprises a sequence set forth in any one of SEQ ID NOs: 62-81.

In certain embodiments, the anti-CD33 VHH CAR comprises a sequence set forth in SEQ ID NO:70 or SEQ ID NO:80.

E. Polypeptides

A variety of polypeptides are contemplated herein including, but not limited to, CD33 VHH DARIC, CD33 VHH DARIC binding component, CD33 DARIC signaling component, anti-CD33 VHH CAR, and fragments thereof. In a preferred embodiment, the polypeptide comprises the amino acid sequence set forth in any one of SEQ ID NOs: 2-82. "Polypeptide", "peptide", and "protein" are used interchangeably according to their conventional meaning, ie, as a sequence of amino acids, unless otherwise indicated. In one embodiment, "polypeptide" includes fusion polypeptides and other variants. Polypeptides can be prepared using any of a variety of well-known recombinant and/or synthetic techniques. Polypeptides are not limited to a particular length, for example, they may include a full-length protein sequence, a fragment of a full-length protein, or a fusion protein, and post-translational modifications of the polypeptide, such as glycosylation, acetylation, phosphorylation, etc. may include other modifications known in the art, including naturally occurring and non-naturally occurring modifications. In particularly preferred embodiments, fusion polypeptides, polypeptides, fragments thereof and other variants are prepared, obtained or isolated from one or more human polypeptides.

As used herein, an “isolated peptide” or “isolated polypeptide” and the like refers to a peptide or polypeptide molecule that has been isolated and/or purified in vitro from the cellular environment and from its associated state with other components of the cell, i.e. in vivo. Refers to a peptide or polypeptide molecule that is not significantly associated with a substance within it. In certain embodiments, the isolated polypeptide is a synthetic polypeptide, a semi-synthetic polypeptide, or a polypeptide obtained or derived from a recombinant source.

Polypeptides include “polypeptide variants”. A polypeptide variant may differ from a naturally occurring polypeptide in one or more substitutions, deletions, additions, and/or insertions. Such variants may occur naturally (eg, splice variants) or may be generated synthetically, eg, by modifying one or more of the above polypeptide sequences. For example, in certain embodiments, it may be desirable to improve the binding affinity and/or other biological properties of a polypeptide by introducing one or more substitutions, deletions, additions, and/or insertions into the polypeptide. In certain embodiments, the polypeptide comprises at least about 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91% , 92%, 93%, 94%, 95%, 86%, 97%, 98%, or 99% amino acid identity, wherein the variant generally retains at least one biological activity of the reference sequence. do. In certain embodiments, the biological activity is binding affinity. In certain embodiments, the biological activity is a cytolytic activity.

Polypeptide variants include biologically active “polypeptide fragments”. Exemplary examples of biologically active polypeptide fragments include an anti-CD33 VHH domain, an intracellular signal transduction domain, and the like. As used herein, the term "biologically active fragment" or "minimally biologically active fragment" refers to at least 100%, at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, at least 40%, at least 30%, at least 20%, at least 10%, or at least 5%. In certain embodiments, a polypeptide fragment may comprise an amino acid chain of at least 5 to about 1700 amino acids in length. In certain embodiments, the fragment is at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700 or more amino acids in length.

In certain embodiments, the polypeptides presented herein may comprise one or more amino acids designated "X" or "Xaa", which are used interchangeably. When “X” occurs in an amino acid SEQ ID NO: it refers to any one or more amino acids. In certain embodiments, the SEQ ID NO: representing a fusion protein comprises a sequence of consecutive X residues that cumulatively represent any amino acid sequence. In certain embodiments, "XX" refers to any two amino acid combination. In certain embodiments, "XX" represents two serines, namely SS. In certain embodiments, "XX" refers to any two amino acid combination that reduces immunogenicity.

In a preferred embodiment, "XX" represents the amino acid KP.

As noted above, polypeptides can be altered in a variety of ways, including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants of a reference polypeptide can be prepared by mutation in DNA. Methods for mutagenesis and nucleotide sequence alteration are well known in the art. See, e.g., Kunkel (1985, Proc. Natl. Acad. Sci. USA. 82: 488-492), Kunkel et al. (1987, Methods in Enzymol, 154: 367-382), USA See Patent No. 4,873,192 to Watson, JD et al. (Molecular Biology of the Gene, Fourth Edition, Benjamin/Cummings, Menlo Park, Calif., 1987) and the references cited therein. Guidelines for appropriate amino acid substitutions that do not affect the biological activity of the protein of interest can be found in the model of Dayhoff et al. (Dayhoff et al. (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found. , Washington, DC]).

In certain embodiments, a polypeptide variant comprises one or more conservative substitutions. A "conservative substitution" is one in which an amino acid is substituted for another amino acid with similar properties, and therefore those skilled in the art of peptide chemistry would expect the secondary structure and hydropathic nature of the polypeptide to be substantially unchanged. Modifications may be modifications in the structure of polynucleotides and polypeptides contemplated in certain embodiments, and may still yield functional molecules encoding variant or derivative polypeptides having the desired properties after modification. When it is desirable to alter the amino acid sequence of a polypeptide to produce an equivalent or even improved variant polypeptide, for example, one of ordinary skill in the art can change one or more of the codons of the coding DNA sequence, e.g. according to Table 1. .

amino acid codon amino acid 1-character code 3-letter code codon alanine A Ala GCA GCC GCG GCU cysteine C Cys UGC UGU aspartic acid D Asp GAC GAU glutamic acid E Glu GAA GAG phenylalanine F Phe UUC UUU glycine G Gly GGA GGC GGG GGU histidine H His CAC CAU isoleucine I iso AUA AUC AUU Lee Sin K Lys AAA AAG leucine L Leu UUA UUG CUA CUC CUG CUU methionine M Met AUG asparagine N Asn AAC AAU proline P Pro CCA CCC CCG CCU glutamine Q Gln CAA CAG arginine R Arg AGA AGG CGA CGC CGG CGU serine S Ser AGC AGU UCA UCC UCG UCU threonine T Thr ACA ACC ACG ACU valine V Val GUA GUC GUG GUU tryptophan W Trp UGG tyrosine Y Tyr UAC UAU

Guidance in determining amino acid residues that can be substituted, inserted, or deleted without impairing biological activity can be found in computer programs well known in the art, for example, DNASTAR, DNA Strider, Geneious, Mac Vector, or Vector NTI software. can be checked using . Preferably, the amino acid changes in the protein variants disclosed herein are conservative amino acid changes, ie substitution of similarly charged or uncharged amino acids. Conservative amino acid changes include the substitution of one of the groups of amino acids related to their side chains. Naturally occurring amino acids are generally divided into four groups: acidic (aspartate, glutamate) amino acids, basic (lysine, arginine, histidine) amino acids, nonpolar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan) amino acids, and uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threon, tyrosine) amino acids. Phenylalanine, tryptophan, and tyrosine are sometimes grouped together as aromatic amino acids. Appropriate conservative substitutions of amino acids in peptides or proteins are known to those skilled in the art and can generally be made without altering the biological activity of the resulting molecule. One of ordinary skill in the art recognizes that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson et al., Molecular Biology of the Gene , 4th Edition, 1987, The Benjamin). /Cummings Pub. Co., p.224).

In one embodiment where expression of two or more polypeptides is desired, the polynucleotide sequences encoding them may be separated by a polynucleotide sequence encoding a ribosome skip sequence or an IRES sequence as disclosed elsewhere herein.

Polypeptides contemplated in certain embodiments include fusion polypeptides. In certain embodiments, fusion polypeptides and polynucleotides encoding the fusion polypeptides are provided. Fusion polypeptides and fusion proteins refer to polypeptides having at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 polypeptide segments. In a preferred embodiment, the fusion polypeptide comprises one or more CD33 VHH DARIC components. In another preferred embodiment, the fusion polypeptide comprises one or more CD33 VHH DARICs.

In another embodiment, two or more CD33 VHH DARIC components and/or other polypeptides may be expressed as a fusion protein comprising one or more self-cleaving peptide sequences between the polypeptides as disclosed elsewhere herein.

In certain embodiments, the fusion polypeptide comprises a CD33 DARIC signal transduction component, a self-cleaving polypeptide sequence or ribosome skip sequence, and a CD33 VHH DARIC binding component.

In certain embodiments, the fusion polypeptide comprises a CD33 DARIC signaling component, a self-cleaving polypeptide sequence or ribosomal skip sequence, a CD33 VHH DARIC binding component, another self-cleaving polypeptide sequence or ribosomal skip sequence, and another target antigen directed against other DARIC binding components.

A fusion polypeptide may comprise: one or more polypeptide domains or segments including, but not limited to, a signal peptide, a cell penetrating peptide domain (CPP), a binding domain, a signal transduction domain, and the like; epitope tags (eg, maltose binding protein (“MBP”), glutathione S transferase (GST), HIS6, MYC, FLAG, V5, VSV-G, and HA); polypeptide linkers; and a polypeptide cleavage signal. Fusion polypeptides are generally C-terminal to N-terminal bonds, but may also be C-terminal to C-terminal bonds, N-terminal to N-terminal bonds, or N-terminal to C-terminal bonds. In certain embodiments, the polypeptides of the fusion protein can be in any order. Fusion polypeptides or fusion proteins may also include conservatively modified variants, polymorphic variants, alleles, mutants, subsequences, and interspecies homologues so long as the desired activity of the fusion polypeptide is preserved. Fusion polypeptides can be produced by chemical synthesis methods, by chemical linkage between two moieties, or generally prepared using other standard techniques. Linked DNA sequences comprising fusion polypeptides are operably linked to appropriate transcriptional or translational regulatory elements as disclosed elsewhere herein.

A fusion polypeptide may optionally include one or more linkers that may be used to join one or more polypeptides or domains within the polypeptide. Peptide linker sequences can be used to separate any two or more polypeptide components by a distance sufficient to ensure that each polypeptide folds into an appropriate secondary and tertiary structure so that the polypeptide domain can exert its desired function. Such peptide linker sequences are incorporated into the fusion polypeptide using standard techniques in the art. Appropriate peptide linker sequences can be selected based on the following factors: (1) the ability to adopt a flexible extended conformation; (2) the inability to adopt secondary structures capable of interacting with functional epitopes on the first and second polypeptides; and (3) lack of hydrophobic or charged residues capable of reacting with a polypeptide functional epitope. In certain embodiments, preferred peptide linker sequences contain Gly, Asn, and Ser residues. Other nearly neutral amino acids such as Thr and Ala may also be used in the linker sequence. Amino acid sequences that can be usefully used as linkers include those described in Maratea et al. ( Gen 40:39-46, 1985); Murphy et al. [ Proc. Natl. Acad. Sci. USA 83:8258-8262, 1986]; U.S. Patent No. 4,935,233, and U.S. Patent No. 4,751,180. A linker sequence is not required if a particular fusion polypeptide segment contains a non-essential N-terminal amino acid region that can be used to separate functional domains and prevent steric interference. In certain embodiments, preferred linkers are flexible amino acid subsequences that are generally synthesized as part of a recombinant fusion protein. A linker polypeptide can be between 1 and 200 amino acids in length, between 1 and 100 amino acids in length, or between 1 and 50 amino acids in length, inclusive of all integer values in between.

Exemplary polypeptide cleavage signals include protease cleavage sites, nuclease cleavage sites (eg, rare restriction enzyme recognition sites, self-cleaving ribozyme recognition sites), and polypeptide cleavage recognition sites such as self-cleaving viral oligopeptides ( see de Felipe and Ryan (2004, Traffic , 5(8); 616-26).

Suitable protease cleavage sites and self-cleaving peptides are known to those skilled in the art (eg, Ryan et al. [1997. J. Gener. Virol. 78, 699-722]; Scymczak et al. (2004) Nature Biotech. 5, 589-594). Exemplary protease cleavage sites include a fortivirus NIa protease (eg, tobacco etch virus protease), a fortivirus HC protease, a fortivirus P1 (P35) protease, a byovirus NIa protease, a bioviral RNA-2-encoded protease, aphthovirus L protease, enterovirus 2A protease, rhinovirus 2A protease, picorna 3C protease, comovirus 24K protease, nepovirus 24K protease, Cleavage sites of RTSV (rice pest globular virus) 3C-like protease, PYVF (Pasnip yellow stain virus) 3C-like protease, heparin, thrombin, factor Xa, and enterokinase. Due to high cleavage stringency, in one embodiment, a TEV (Tobacco Etching Virus) protease cleavage site, for example, EXXYXQ(G/S) (SEQ ID NO: 95) such as ENLYFQG (SEQ ID NO: 96) and ENLYFQS (SEQ ID NO: 97) ), wherein X represents any amino acid (cleavage by TEV occurs between Q and G or between Q and S).

In certain embodiments, the polypeptide cleavage signal is a viral self-cleaving peptide or a ribosome skipping sequence.

Illustrative examples of ribosome skipping sequences include, but are not limited to, 2A or 2A-like sites, sequences, or domains (Donnelly et al., 2001. J. Gen. Virol . 82:1027-1041). Reference). In certain embodiments, the viral 2A peptide is an aphtovirus 2A peptide, a fortivirus 2A peptide, or a cardiovirus 2A peptide.

In one embodiment, the viral 2A peptide is selected from the group consisting of: foot-and-mouth disease virus (FMDV) 2A peptide, equine rhinitis A virus (ERAV) 2A peptide, tosea sub Thosea asigna virus, TaV) 2A peptide, porcine tescovirus-1 (PTV-1) 2A peptide, Theilovirus 2A peptide, and encephalomyocarditis virus 2A peptide.

Illustrative examples of site 2A are provided in Table 2.

SEQ ID NO: 98 GSGATNFSLLKQAGDVEENPGP SEQ ID NO: 99 ATNFSLLKQAGDVEENPGP SEQ ID NO: 100 LLKQAGDVEENPGP SEQ ID NO: 101 GSGEGRGSLLTCGDVEENPGP SEQ ID NO: 102 EGRGSLLTCGDVEENPGP SEQ ID NO: 103 LLTCGDVEENPGP SEQ ID NO: 104 GSGQCTNYALLKLAGDVESNPGP SEQ ID NO: 105 QCTNYALLKLAGDVESNPGP SEQ ID NO: 106 LLKLAGDVESNPGP SEQ ID NO: 107 GSGVKQTLNFDLLKLAGDVESNPGP SEQ ID NO: 108 VKQTLNFDLLKLAGDVESNPGP SEQ ID NO: 109 LLKLAGDVESNPGP SEQ ID NO: 110 LLNFDLLKLAGDVESNPGP SEQ ID NO: 111 TLNFDLLKLAGDVESNPGP SEQ ID NO: 112 LLKLAGDVESNPGP SEQ ID NO: 113 NFDLLKLAGDVESNPGP SEQ ID NO: 114 QLLNFDLLKLAGDVESNPGP SEQ ID NO: 115 APVKQTLNFDLLKLAGDVESNPGP SEQ ID NO: 116 VTELLYRMKRAETYCPRPLLAIHPTEARHKQKIVAPVKQT SEQ ID NO: 117 LNFDLLKLAGDVESNPGP SEQ ID NO: 118 LLAIHPTEARHKQKIVAPVKQTLNFDLLKLAGDVESNPGP SEQ ID NO: 119 EARHKQKIVAPVKQTLNFDLLKLAGDVESNPGP

In a preferred embodiment, the polypeptide or fusion polypeptide comprises one or more CD33 DARIC components, CD33 VHH DARIC, or anti-CD33 VHH CAR.

In a preferred embodiment, the fusion polypeptide comprises a CD33 DARIC signaling component and a CD33 VHH DARIC binding component separated by a self-cleaving polypeptide sequence.

In certain embodiments, the fusion polypeptide comprises a sequence set forth in any one of SEQ ID NOs: 32-61. In certain embodiments, the fusion polypeptide comprises a sequence set forth in any one of SEQ ID NOs: 40, 50, or 60.

In certain embodiments, the fusion polypeptide comprises a CD33 DARIC signaling component; viral self-cleaving 2A polypeptide; and a CD33 VHH DARIC binding component, wherein the CD33 DARIC signaling component comprises a FRB T2098L multimerization domain, a CD8α transmembrane domain, a CD137 costimulatory domain, and a CD3ζ primary signaling domain, wherein the CD33 VHH DARIC binding component is an anti- CD33 VHH, FKBP12 multimerization domain polypeptide, and CD4 transmembrane domain.

In certain embodiments, the fusion polypeptide comprises a sequence set forth in any one of SEQ ID NOs: 32-41. In certain embodiments, the fusion polypeptide comprises the sequence set forth in SEQ ID NO:40.

In certain embodiments, the fusion polypeptide comprises a CD33 DARIC signaling component comprising a FRB T2098L multimerization domain, a CD8α transmembrane domain, a CD137 costimulatory domain, and a CD3ζ primary signaling domain; viral self-cleaving 2A polypeptide; and an anti-CD33 VHH, CD4 transmembrane domain, optionally comprising a CD27, CD28, TNFRS14, TNFRS18, TNFRS25, OX40, or TNFR2 costimulatory domain.

In certain embodiments, the fusion polypeptide comprises a sequence set forth in any one of SEQ ID NOs: 42-51. In certain embodiments, the fusion polypeptide comprises the sequence set forth in SEQ ID NO:50.

In certain embodiments, the fusion polypeptide comprises a sequence set forth in any one of SEQ ID NOs: 52-61. In certain embodiments, the fusion polypeptide comprises the sequence set forth in SEQ ID NO:60.

F. Polynucleotides

In certain embodiments, polynucleotides encoding CD33 VHH DARIC, CD33 VHH DARIC binding component, CD33 DARIC signaling component, anti-CD33 VHH CAR, and fragments thereof are provided. As used herein, the term “polynucleotide” or “nucleic acid” refers to deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and DNA/RNA hybrids. Polynucleotides can be single-stranded or double-stranded and can be recombinant, synthetic, or isolated. Polynucleotides include, but are not limited to: precursor messenger RNA (pre-mRNA), messenger RNA (mRNA), RNA, synthetic RNA, synthetic mRNA, genomic DNA (gDNA), PCR amplified DNA, complementary DNA (cDNA), synthetic DNA, or recombinant DNA. polynucleotides are at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 100, at least 200, at least 300, at least 400 , at least 500, at least 1000, at least 5000, at least 10000, or at least 15000, or more (including any intermediate length) nucleotides (ribonucleotides or deoxyribonucleotides, or any one nucleotide) modified form) refers to a multimeric form of nucleotides. In this context, “intermediate length” means any length between recited values, such as 6, 7, 8, 9, etc.; 101, 102, 103, etc.; 151, 152, 153, etc.; It will be readily understood to mean 201, 202, 203, etc. In certain embodiments, the polynucleotide or variant is at least or about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, relative to the reference sequence; 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93% , 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity.

As used herein, "isolated polynucleotide" refers to a polynucleotide that has been purified from a sequence flanking it in its naturally occurring state, eg, a DNA fragment that has been removed from a sequence generally adjacent to the fragment. In certain embodiments, "isolated polynucleotide" also refers to complementary DNA (cDNA), recombinant DNA, or other polynucleotides that do not exist in nature and are made by human hands. In certain embodiments, an isolated polynucleotide is a synthetic polynucleotide, a semi-synthetic polynucleotide, or a polynucleotide obtained or derived from a recombinant source.

In various embodiments, a polynucleotide comprises an mRNA encoding a polynucleotide contemplated herein. In certain embodiments, the mRNA comprises a cap, one or more nucleotides, and a poly(A) tail.

In certain embodiments, polynucleotides encoding one or more CD33 VHH DARIC components may be codon optimized. As used herein, the term “codon-optimized” refers to substituting codons in a polynucleotide encoding a polypeptide to increase the expression, stability, and/or activity of the polypeptide. Factors influencing codon optimization include, but are not limited to, one or more of the following: (i) a bias table constructed as a synthesis or variation of codon bias between two or more organisms or genes; (ii) variations in the degree of codon bias within an organism, gene, or set of genes; (iii) systematic variation of codons including context; (iv) codon change according to the coding tRNA of the codon; (v) codon variation as a function of percent GC in all or one of the triplets; (vi) variations in the degree of similarity to a reference sequence, such as a naturally occurring sequence; (vii) variation in codon frequency cutoff; (viii) structural properties of mRNA transcribed from the DNA sequence; (ix) prior knowledge of the function of the DNA sequence underlying the codon substitution set design; (x) systematic variation of the codon set for each amino acid; and/or (xi) isolated removal of a false translation initiation site.

As used herein, the term “nucleotide” refers to a heterocyclic nitrogen base N-glycosidically linked to a phosphorylated sugar. Nucleotides are understood to include natural bases as well as a wide variety of modified bases recognized in the art. Such bases are generally located at the 1' position of the moiety per nucleotide. Nucleotides generally include bases, sugars, and phosphate groups. The sugar in ribonucleic acid (RNA) is ribose, and the sugar in deoxyribonucleic acid (DNA) is deoxyribose, that is, a sugar lacking the hydroxyl group present in ribose.

Illustrative examples of polynucleotides include, but are not limited to, polynucleotides encoding the polypeptides set forth in SEQ ID NOs: 2-82.

In various exemplary embodiments, a polynucleotide contemplated herein comprises one or more CD33 VHH DARIC components, a CD33 VHH DARIC receptor, an anti-CD33 VHH CAR, a polynucleotide encoding a fusion polypeptide, and a polynucleotide contemplated herein. expression vectors, viral vectors, and transfer plasmids.

As used herein, the terms "polynucleotide variant" and "variant" and the like refer to a polynucleotide that exhibits substantial sequence identity to a reference polynucleotide sequence, or a polynucleotide that hybridizes to a reference sequence under stringent conditions as defined below. . These terms also include polynucleotides that are distinguished from a reference polynucleotide by addition, deletion, substitution, or modification of at least one nucleotide. Accordingly, the terms "polynucleotide variant" and "variant" include polynucleotides in which one or more nucleotides have been added or deleted, modified, or substituted with different nucleotides. In this regard, it is well understood in the art that certain alterations, including mutations, additions, deletions, and substitutions, can be made to a reference polynucleotide, whereby the altered polynucleotide retains the biological function or activity of the reference polynucleotide. do.

As used herein, terms comprising "sequence identity" or, e.g., "sequences that are 50% identical" refer to the degree to which sequences are identical on a nucleotide-to-nucleotide basis or on an amino acid-to-amino acid basis over a comparison window. refers to Thus, "percentage of sequence identity" compares two sequences that are optimally aligned on a comparison window; Identical nucleic acid bases (eg A, T, C, G, I) or identical amino acid residues (eg Ala, Pro, Ser, Thr, Gly, Val, Leu, He, Phe, Tyr, Trp, Lys, Arg, His , Asp, GLu, Asn, Gln, Cys, and Met) determine the number of positions occurring in both sequences to obtain the number of matching positions; divide the number of matched positions by the total number of positions within the comparison window (ie, window size); It can be calculated by multiplying the result by 100 to get the percent sequence identity. Any one of the reference sequences described herein and at least about 50%, 55%, 60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74% , 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 86%, 97%, 98%, or 99% sequence identity.

Polynucleotides contemplated herein, irrespective of the length of the coding sequence itself, include other DNA sequences, such as promoters and/or enhancers, untranslated regions ( UTR), signal sequence, Kozak sequence, polyadenylation signal, additional restriction enzyme sites, multiple cloning sites, internal ribosome entry sites (IRES), recombinase recognition sites (eg LoxP, FRT, and Att sites), stop codons, Because they can be combined with transcription termination signals, and polynucleotides encoding self-cleaving polypeptides, epitope tags, their overall length can vary significantly. Accordingly, it is contemplated that polynucleotide fragments of almost any length may be used, and the overall length may preferably be limited by ease of manufacture and ease of use in the intended recombinant DNA protocol.

Polynucleotides can be prepared, manipulated, expressed, and/or delivered using any of a variety of well-established techniques known and available in the art. To express the desired polypeptide, a nucleotide sequence encoding the polypeptide can be inserted into an appropriate vector.

Illustrative examples of vectors include, but are not limited to, plasmids, autonomously replicating sequences, and transposable elements such as transgenic elements such as Sleeping Beauty, PiggyBac.

Further illustrative examples of vectors include plasmids, phagemids, cosmids, artificial chromosomes (such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC), or P1-derived artificial chromosome (PAC)), bacteriophages (such as lambda). phage or M13 phage), and animal viruses.

Illustrative examples of viruses useful as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (eg, herpes simplex virus), varicellaviruses, baculoviruses, papillomaviruses, and papovaviruses. (eg SV40).

Illustrative examples of expression vectors include the pClneo vector (Promega) for expression in mammalian cells; pLenti4/V5-DEST®, pLenti6/V5-DEST®, and pLenti6.2/V5-GW/lacZ (Invitrogen) for lentivirus-mediated gene delivery and expression in mammalian cells. . In certain embodiments, the coding sequence of a polypeptide disclosed herein can be linked to such an expression vector for expression of the polypeptide in mammalian cells.

In certain embodiments, the vector is an episomal vector or an extrachromosomally maintained vector. As used herein, the term “episomal” refers to a vector capable of replication without integration into the host's chromosomal DNA and capable of replication without progressive loss from a dividing host cell, wherein the vector is capable of replication either extrachromosomally or It also means that it replicates in the episome.

"Expression control sequences", "regulatory elements", or "regulatory sequences" present in an expression vector are the untranslated regions of the vector, including the origin of replication, selection cassette, promoter, enhancer, translation initiation signal (Shine Dalgarno sequence or Kozak sequence) introns, polyadenylation sequences, 5' and 3' untranslated regions, all of which interact with host cell proteins to effect transcription and translation. These factors may vary in strength and specificity. Depending on the vector system and host used, any number of suitable transcriptional and translational elements can be used, including ubiquitous and inducible promoters.

In certain embodiments, polynucleotides include vectors including but not limited to expression vectors and viral vectors. A vector may include one or more exogenous, endogenous, or heterologous regulatory sequences, such as promoters and/or enhancers. An “endogenous regulatory sequence” is a sequence that is naturally associated with a given gene in a genome. An “exogenous regulatory sequence” is a sequence that is juxtaposed with a gene by genetic manipulation (ie, molecular biology techniques) such that the transcription of the gene is driven by the linked enhancer/promoter. A “heterologous regulatory sequence” is an exogenous sequence derived from a species different from the genetically engineered cell. "Synthetic" regulatory sequences may include elements of one or more endogenous and/or exogenous sequences, and/or sequences determined in vitro or in a virtual environment to provide optimal promoter and/or enhancer activity for a particular therapy.

As used herein, the term “promoter” refers to the recognition site of a polynucleotide (DNA or RNA) to which RNA polymerase binds. RNA polymerase initiates and transcribes a polynucleotide operably linked to a promoter. In certain embodiments, a promoter operating in mammalian cells comprises an AT-rich region located approximately 25 to 30 bases upstream from the site where transcription is initiated and/or a CNCAAT region located 70 to 80 bases upstream from the transcription initiation region. (wherein N can be any nucleotide).

The term "enhancer" refers to a segment of DNA that contains sequences capable of enhancing transcription, which in some cases can act independently of orientation relative to other regulatory sequences. Enhancers may act cooperatively or additionally with promoters and/or other enhancer elements. The term “promoter/enhancer” refers to a segment of DNA that contains a sequence capable of providing both promoter and enhancer function.

The term “operably linked” refers to a juxtaposition in which the described components are in a relationship that enables them to function in their intended manner. In one embodiment, the term refers to a functional linkage between a nucleic acid expression control sequence (eg, a promoter and/or enhancer) and a second polynucleotide sequence (eg, a polynucleotide of interest), wherein the expression control sequence is a second Inducing transcription of a nucleic acid corresponding to the sequence.

As used herein, the term “constitutive expression control sequence” refers to a promoter, enhancer, or promoter/enhancer that continues or continuously enables transcription of an operably linked sequence. Constitutive expression control sequences are “ubiquitous” promoters, enhancers, or promoters/enhancers that allow expression in a wide variety of cell and tissue types, or are “cell-specific” that allow expression in each of a limited range of cells and tissue types. ", "cell type specific", "cell lineage specific", or "tissue specific" promoter, enhancer, or promoter/enhancer.

Exemplary ubiquitous expression control sequences suitable for use in certain embodiments include, but are not limited to: cytomegalovirus (CMV) early early promoter, viral simian virus 40 (SV40) (eg, early or late) , murine murine leukemia virus (MoMLV) LTR promoter, roux sarcoma virus (RSV) LTR, herpes simplex virus (HSV) (thymidine kinase) promoter, H5, P7.5, and P11 promoters derived from vaccinia virus, elongation Factor 1-alpha (EF1a) promoter, early growth response 1 (EGR1), ferritin H (FerH), ferritin L (FerL), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), eukaryotic translation initiation factor 4A1 (EIF4A1) , heat shock 70 kDa protein 5 (HSPA5), heat shock protein 90 kDa beta member 1 (HSP90B1), heat shock protein 70 kDa (HSP70), β-kinesin (β-KIN), human ROSA 26 locus (Irions et al. Nature Biotechnology 25, 1477 - 1482 (2007)), ubiquitin C promoter (UBC), phosphoglycerate kinase-1 (PGK) promoter, cytomegalovirus enhancer/chicken β-actin (CAG) promoter, β-actin promoter and myeloproliferative The sarcoma virus enhancer, negative control region, is deleted and the d1587rev primer binding site is substituted for the (MND) U3 promoter (see Haas et al., Journal of Virology. 2003;77(17):9439-9450).

In one embodiment, the vector comprises a MNDU3 promoter.

In one embodiment, the vector comprises an EF1a promoter comprising the first intron of the human EF1a gene.

In one embodiment, the vector comprises an EF1a promoter lacking the first intron of the human EF1a gene.

In certain embodiments, cell, cell type, cell lineage, or tissue specific expression control sequences are used to achieve cell type specific, lineage specific, or tissue specific expression of a desired polynucleotide sequence (e.g., It may be desirable to express a particular nucleic acid encoding a polypeptide only in a subgroup of a cell type, cell lineage, or tissue, or during a particular stage of development.

In certain embodiments, it may be desirable to express the polynucleotide as a T cell specific promoter.

As used herein, “conditional expression” refers to inducible expression; inhibitory expression; It may refer to any type of conditional expression, including, but not limited to, expression in a cell or tissue having a particular physiological, biological, or disease state, and the like. This definition is not intended to exclude cell type or tissue specific expression. Certain embodiments provide for conditional expression of a polynucleotide of interest, For example, expression is regulated by subjecting a cell, tissue, organism, etc. to a treatment or condition that causes expression of a polynucleotide or causes an increase or decrease in expression of a polynucleotide encoded by a polynucleotide of interest. do.

Exemplary examples of inducible promoters/systems include steroid-inducible promoters, such as promoters for genes encoding glucocorticoid or estrogen receptors (inducible by treatment with such hormones), metallotionine promoters (treatment with various heavy metals). ( inducible by WO 2002/088346), a tetracycline dependent control system, and the like. Inducing agents include, but are not limited to, glucocorticoids, estrogens, mifepristone (RU486), metals, interferons, small molecules, cuminates, tetracyclines, doxycyclines, and variants thereof.

As used herein, an “internal ribosome entry site” or “IRES” is one that promotes direct internal ribosome entry to an initiation codon, such as ATG, of a cistron (protein coding region) leading to cap-independent translation of a gene. refers to the element. For example, Jackson et al. [1990. Trends Biochem Sci 15(12):477-83) and Jackson and Kaminski, 1995. RNA 1(10):985-1000]. Examples of IRESs commonly used by those skilled in the art include those described in US Pat. No. 6,692,736. Additional examples of "IRES" known in the art include IRES obtainable from picornavirus (Jackson et al. 1990), and, for example, immunoglobulin heavy chain binding protein (BiP), vascular endothelial growth factor (VEGF) ( See Huez et al. (1998. Mol. Cell. Biol. 18(11):6178-6190), fibroblast growth factor 2 (FGF-2), and insulin-like growth factor (IGFII), translation initiation factor eIF4G , and yeast transcription factors TFIID and HAP4, a source of viral or cellular mRNA such as Encephalitis Virus (EMCV) commercially available from Novagen (see Duke et al., 1992. J. Virol 66(3):1602-9). IRES obtainable from, and VEGF IRES (see Huez et al., 1998. Mol Cell Biol 18(11):6178-90). IRESs have also been reported in the viral genomes of picornavirus, dicistrovirus and flavivirus species, and in HCV, Friends murine leukemia virus (FrMLV), and Moloney murine leukemia virus (MoMLV).

In one embodiment, the IRES used in the polynucleotides contemplated herein is an EMCV IRES.

In certain embodiments, the polynucleotides comprise a consensus Kozak sequence. As used herein, the term “Kozak sequence” refers to a short nucleotide sequence that significantly facilitates initial binding of mRNA to the small subunit of the ribosome and increases translation. The consensus Kozak sequence is (GCC)RCCATGG (SEQ ID NO:83), where R is a purine (A or G) (Kozak, 1986. Cell . 44(2):283-92) and Kozak, 1987. Nucleic Acids Res . 15(20):8125-48).

Factors that induce efficient termination and polyadenylation of heterologous nucleic acid transcripts increase heterologous gene expression. A transcription termination signal is generally identified downstream of the polyadenylation signal. In certain embodiments, the vector comprises a polyadenylation sequence 3' to the polynucleotide encoding the polypeptide to be expressed. As used herein, the term “polyA site” or “polyA sequence” refers to a DNA sequence that directs both polyadenylation and termination of initial RNA transcription by RNA polymerase II. The polyadenylation sequence can promote mRNA stability by adding a polyA tail to the 3' end of the coding sequence, thereby contributing to an increase in translation efficiency. Cleavage and polyadenylation are driven by the poly(A) sequence in the RNA. The core poly(A) sequence for mammalian mRNA precursors has two recognition elements flanked by cleavage-polyadenylation sites. In general, the nearly constant AAUAAA hexamers lie 20 to 50 nucleotides upstream from the more variable elements rich in U or GU residues. Cleavage of the initial transcript occurs between these two elements and binds up to 250 adenosines added to the 5' cleavage product. In certain embodiments, the core poly(A) sequence is an ideal polyA sequence (eg, AATAAA, ATTAAA, AGTAAA). In certain embodiments, the poly(A) sequence comprises a SV40 polyA sequence, a bovine growth hormone polyA sequence (BGHpA), a rabbit β-globin polyA sequence (rβgpA), a variant thereof, or other suitable heterologous sequence known in the art. or an endogenous polyA sequence. In certain embodiments, the poly(A) sequence is a synthetic sequence.

In certain embodiments, polynucleotides encoding one or more polypeptides or fusion polypeptides can be introduced into immune effector cells (eg, T cells) by both nonviral and viral methods. In certain embodiments, delivery of one or more polynucleotides may be provided by the same method or different methods and/or may be provided by the same vector or different vectors.

The term “vector” is used herein to refer to a nucleic acid molecule capable of delivering or transporting another nucleic acid molecule. The delivered nucleic acid is generally linked to a vector nucleic acid molecule, eg, inserted into a vector nucleic acid molecule. A vector may contain sequences that induce autonomous replication in a cell, or may contain sequences sufficient to permit integration into host cell DNA. In certain embodiments, non-viral vectors are used to deliver one or more polynucleotides contemplated herein to a T cell.

Illustrative examples of non-viral vectors include, but are not limited to, plasmids (eg, DNA plasmids or RNA plasmids), transposons, cosmids, and bacterial artificial chromosomes.

Exemplary methods of non-viral delivery of polynucleotides contemplated in certain embodiments include: electroporation, sonication, lipofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, nanoparticles, polycations or lipid:nucleic acid conjugates, naked DNA, artificial virions, DEAE-dextran mediated delivery, gene guns, and heat-shock.

Exemplary embodiments of polynucleotide delivery systems suitable for use in certain embodiments contemplated in certain embodiments include, but are not limited to, those provided by Amaxa Biosystems, Maxcyte, Inc., BTX Molecular Delivery Systems, and Copernicus Therapeutics Inc. it doesn't happen Lipofection reagents are sold commercially (eg Transfectam® and Lipofectin®). Cationic and neutral lipids suitable for efficient receptor recognition lipofection of polynucleotides have been described in the literature. See, eg, Liu et al. (2003) Gene Therapy. 10:180-187]; and Balazs et al. (2011) Journal of Drug Delivery. 2011:1-12]. Antibody-targeted, bacterial-derived, in vivo nanocell-based delivery is also contemplated in certain embodiments.

Viral vectors comprising polynucleotides contemplated in certain embodiments are, as described below, generally by systemic administration (eg, intravenous, intraperitoneal, intramuscular, subcutaneous, or intracranial injection) or topical application. It can be delivered in vivo by administration to an individual patient. Alternatively, the vector can be delivered ex vivo to cells explanted from an individual patient (eg, mobilized peripheral blood, lymphocytes, bone marrow aspiration, tissue biopsy, etc.) or cells such as universal donor hematopoietic stem cells, followed by a corresponding The cells can be re-implanted into the patient.

In one embodiment, a viral vector comprising a polynucleotide contemplated herein is administered directly to an organism for transduction of cells in vivo. Alternatively, naked DNA may be administered. Administration is by any one of the routes commonly used to introduce molecules into eventual contact with blood or tissue cells, including but not limited to injection, infusion, topical application, and electroporation. Appropriate methods of administering such nucleic acids are available and well known to those skilled in the art, and although more than one route may be used to administer a particular composition, one route is often capable of providing a more immediate and more effective response than another route.

Exemplary embodiments of viral vector systems suitable for use in certain embodiments contemplated in certain embodiments include, but are not limited to, adeno-associated virus (AAV), retrovirus, herpes simplex virus, adenovirus, and vaccinia virus vectors. not limited

In various embodiments, one or more polynucleotides encoding one or more CD33 VHH DARIC components and/or other polypeptides contemplated herein are recombinant comprising said one or more polynucleotides. Adeno-associated virus (rAAV) is introduced into immune effector cells, eg, T cells, by transducing the cells into the cells.

AAV is a small (about 26 nm) replication-defective, predominantly episomal, enveloped virus. AAV can infect both dividing and non-dividing cells and incorporate its genome into the genome of the host cell. Recombinant AAV (rAAV) generally consists of at least a transgene and its regulatory sequences, and 5' and 3' AAV inverted terminal repeats (ITRs). The ITR sequence is about 145 bp in length. In certain embodiments, the rAAV comprises an ITR sequence and a capsid sequence isolated from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10.

In some embodiments, a chimeric rAAV is used, wherein the ITR sequence is isolated from one AAV serotype and the capsid sequence is isolated from a different AAV serotype. For example, a rAAV having an ITR sequence from AAV2 and a capsid sequence from AAV6 is referred to as AAV2/AAV6. In certain embodiments, the rAAV vector may comprise an ITR derived from AAV2 and a capsid protein isolated from any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10. In a preferred embodiment, the rAAV comprises an ITR sequence derived from AAV2 and a capsid sequence derived from AAV6. In a preferred embodiment, the rAAV comprises an ITR sequence derived from AAV2 and a capsid sequence derived from AAV2.

In some embodiments, engineering and selection methods can be applied to AAV capsids to increase their likelihood of transducing cells of interest.

Construction of rAAV vectors, their production and purification are described, for example, in US Pat. Nos. 9,169,494; 9,169,492; 9,012,224; 8,889,641; 8,809,058; and 8,784,799, each of which is incorporated herein by reference in its entirety.

In various embodiments, one or more polynucleotides encoding one or more CD33 VHH DARIC components and/or other polypeptides contemplated herein transduce a retrovirus (eg, a lentivirus) comprising the one or more polynucleotides into a cell. thereby being introduced into immune effector cells, eg, T cells.

As used herein, the term “retrovirus” refers to an RNA virus that reverse transcribes its genomic RNA into a linear double-stranded DNA copy and then covalently integrates its genomic DNA into the host genome. Exemplary retroviruses suitable for use in certain embodiments include, but are not limited to: Moloney murine leukemia virus (M-MuLV), Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma Virus (HaMuSV), murine mammary tumor virus (MuMTV), gibbon simian leukemia virus (GaLV), feline leukemia virus (FLV), spumavirus, friend murine leukemia virus, murine stem cell virus (MSCV) , and Rous sarcoma virus (RSV), and lentivirus.

As used herein, the term “lentivirus” refers to a group (or genus) of complex retroviruses. Exemplary lentiviruses include HIV (human immunodeficiency virus; including HIV type 1 and HIV type 2); visna-maedi virus (VMV); caprin arthritis-encephalitis virus (CAVAV); Equine Infectious Anemia Virus (EIAV); feline immunodeficiency virus (FIV); bovine immunodeficiency virus (BIV); and simian immunodeficiency virus (SIV). In one embodiment, HIV based vector backbones (ie, HIV cis-acting sequence elements) are preferred.

In various embodiments, the lentiviral vectors contemplated herein comprise one or more LTRs, and one or more or all of the following accessory elements: cPPT/FLAP, cy (ø) packaging signal, export element, poly(A) sequence , optionally a WPRE or HPRE as discussed elsewhere herein, an insulator element, a selectable marker, and an apoptosis gene.

In certain embodiments, the lentiviral vectors contemplated herein may be integral or non-integrating or integration defective lentiviruses. As used herein, the term “integration defective lentivirus (IDLV)” refers to a lentivirus having an integrase that lacks the ability to integrate the viral genome into the genome of a host cell. An integration-deficient viral vector is described in patent application WO 2006/010834, which is incorporated herein by reference in its entirety.

Exemplary mutations in the HIV-1 pole gene suitable for reducing integrase activity include, but are not limited to: H12N, H12C, H16C, H16V, S81 R, D41A, K42A, H51A, Q53C, D55V , D64E, D64V, E69A, K71A, E85A, E87A, D116N, D1161, D116A, N120G, N1201, N120E, E152G, E152A, D35E, K156E, K156A, E157A, K159E, K159A, K160A, R166A, D167A, E170A, D167A, E170A , K173A, K186Q, K186T, K188T, E198A, R199c, R199T, R199A, D202A, K211A, Q214L, Q216L, Q221 L, W235F, W235E, K236S, K236A, K246A, G247W, D253A, R262A, R263A, and K264H.

The term "long terminal repeat (LTR)" refers to a domain of base pairs located at the ends of retroviral DNA, which, in the context of their natural sequence, is a direct repeat and the U3, R, and U5 regions. contains

As used herein, the term "FLAP element" or "cPPT/FLAP" refers to a retrovirus, such as HIV-1 or HIV-2, comprising the central polypurine pathway and central termination sequences (cPPT and CTS). Refers to a nucleic acid having a sequence. Suitable FLAP elements are described in US Pat. No. 6,682,907 and in Zennou et al., 2000, Cell , 101:173.

As used herein, the term “packaging signal” or “packaging sequence” refers to a cyto[ø] sequence located within the retroviral genome, which is required for insertion of viral RNA into a viral capsid or particle. For example, Clever et al. [1995. J. of Virology , Vol. 69, No. 4; pp. 2101-2109].

The term “export element” refers to a cis-acting post-transcriptional regulatory element that regulates the transport of RNA transcripts from the nucleus to the cytoplasm of a cell. Examples of RNA export elements include human immunodeficiency virus (HIV) rev response element (RRE) (eg, Cullen et al. [1991. J. Virol . 65: 1053]; and Cullen et al. [1991. Cell 58] : 423), and the hepatitis B virus post-transcriptional regulatory element (HPRE).

In certain embodiments, expression of a heterologous sequence in a viral vector is increased by incorporating into the vector a post-transcriptional regulatory element, an efficient polyadenylation site, and optionally a transcription termination signal. Woodchuck hepatitis virus post-transcriptional regulatory elements (WPRE; see Zufferey et al., 1999, J. Virol ., 73:2886); Various post-transcriptional regulatory elements, such as post-transcriptional regulatory elements present in hepatitis B virus (HPRE) (see Huang et al. [ Mol. Cell. Biol ., 5:3864]), and the like, increase the expression of heterologous nucleic acids in proteins. (see Liu et al. [1995, Genes Dev ., 9:1766]).

The lentiviral vector preferably contains several safety enhancing substances as a result of the modification of the LTR. A “self-inactivation (SIN)” vector is such that the right (3′) LTR enhancer-promoter region, known as the U3 region, prevents excessive viral transcription after one round of viral replication (e.g., deletion or refers to a replication-defective vector that has been modified (by substitution), eg, a retroviral or lentiviral vector. Self inactivation is preferably achieved by introducing a deletion in the U3 region of the 3' LTR of the vector DNA, ie the DNA used to produce the vector RNA. Thus, during reverse transcription, this deletion is transferred to the 5' LTR of the proviral DNA. In certain embodiments, it is desirable to greatly reduce or eliminate the production of full-length vector RNA in transduced cells by removing sufficient U3 sequence to significantly reduce or completely eliminate the transcriptional activity of the LTR. In the case of HIV-based lentivectors, these vectors were found to be resistant to significant U3 deletions, including deletion of the LTR TATA box (eg, deletion of -418 to -18) without significant reduction in vector titer. .

An additional safety enhancement is provided by substituting a heterologous promoter for the U3 region of the 5' LTR to induce transcription of the viral genome during viral particle production. Examples of heterologous promoters that may be used include, for example, virus simian virus 40 (SV40) (eg, early or late), cytomegalovirus (CMV) (eg, early), Moloney murine leukemia. Virus (MoMLV), Rous Sarcoma Virus (RSV), and Herpes Simplex Virus (HSV) (thymidine kinase) promoters.

As used herein, the term “pseudotype” or “pseudotyping” refers to a virus in which a viral envelope protein is substituted for a protein of another virus with desirable properties. For example, HIV can be pseudotyped with the vesicular stomatitis virus G-protein (VSV-G) envelope protein, in which the HIV envelope protein (encoded by the env gene) normally targets the virus to CD4 ⁺ presenting cells. This allows HIV to infect a wider range of cells.

In certain embodiments, lentiviral vectors are produced according to known methods. For example, Kutner et al. [ BMC Biotechnol. 2009;9:10. doi: 10.1186/1472-6750-9-10]; Kutner et al. [ Nat. Protoc. 2009;4(4):495-505. doi: 10.1038/nprot.2009.22].

According to certain embodiments contemplated herein, most or all of the viral vector backbone sequences are derived from a lentivirus, eg , HIV-1. However, many different sources of retroviral and/or lentiviral sequences can be used or combined, and numerous substitutions and alterations in specific lentiviral sequences can be accommodated without compromising the ability of the transfer vector to perform the functions described herein. You have to understand that it can be. In addition, a variety of lentiviral vectors are known in the art and are described by Naldini et al. [1996a, 1996b, and 1998]; Zuffery et al. [1997]; Dull et al. [1998], US Pat. No. 6,013,516; and 5,994,136, many of which may be suitable for producing the viral vectors or transfer plasmids contemplated herein.

In various embodiments, one or more polynucleotides encoding one or more CD33 VHH DARIC components and/or other polypeptides contemplated herein are introduced into an immune effector cell by transducing the cell with an adenovirus comprising said one or more polynucleotides. do.

Adenovirus-based vectors can be transduced with very high efficiency in many cell types and do not require cell division. With this vector, high titers and high levels of expression were obtained. Such vectors can be produced in large quantities in a relatively simple system. Most adenoviral vectors are engineered such that the transgene replaces the Ad E1a, E1b, and/or E3 genes; Subsequently, the replication defective vector is propagated in human 293 cells supplying the gene function deleted in trans. Ad vectors are capable of transducing many types of tissue in vivo, including non-dividing, differentiated cells such as those found in liver, kidney, and muscle. Conventional Ad vectors have a large loading capacity.

A native helper cell line designated 293 can be used for the generation and propagation of current replication-deficient adenoviral vectors, which have been transformed from human embryonic kidney cells by an Ad5 DNA fragment and constitutively express the E1 protein (Graham et al.). of 1977 literature). Because the E3 region can be partitioned from the adenoviral genome (Jones & Shenk, 1978), current adenoviral vectors carry foreign DNA in the E1 region, the D3 region, or both with the aid of 293 cells (Graham). & Prevec, 1991). Adenoviral vectors have been used for eukaryotic gene expression (Levrero et al., 1991; Gomez-Foix et al., 1992) and vaccine development (Grunhaus & Horwitz, 1992; Graham & Prevec, 1992). Studies of administering recombinant adenovirus to different tissues include tracheal instillation (Rosenfeld et al. 1991; Rosenfeld et al. 1992), intramuscular injection (Ragot et al. 1993), peripheral intravenous injection (Herz & Gerard et al. 1993), and stereotactic injection into the brain. Inoculation (Le Gal La Salle et al. 1993). Examples of the use of Ad vectors in clinical trials included polynucleotide therapy for antitumor immunization by intramuscular injection (see Sterman et al., Hum. Gene Ther. 7:1083-9 (1998)).

In various embodiments, the one or more polynucleotides encoding one or more CD33 VHH DARIC components and/or other polypeptides contemplated herein comprises a herpes simplex virus (eg, HSV-1, HSV-2) comprising one or more polynucleotides. It is introduced into an immune effector cell by transducing the cell.

Mature HSV virions consist of an enveloped icosahedral capsid with a viral genome consisting of a 152 kb linear double-stranded DNA molecule. In one embodiment, the HSV-based viral vector lacks one or more essential or non-essential HSV genes. In one embodiment, the HSV-based viral vector is replication deficient. Most replication deficient HSV vectors contain deletions to remove one or more mid-early, early or late HSV genes to prevent replication. For example, the HSV vector may lack a very early gene selected from the group consisting of: ICP4, ICP22, ICP27, ICP47, and combinations thereof. Advantages of HSV vectors are their ability to enter a latent phase, which can result in long-term expression of DNA, and their large viral DNA genome, which can accommodate exogenous DNA inserts of up to 25 kb. HSV-based vectors are described, for example, in US Pat. Nos. 5,837,532, 5,846,782, and 5,804,413, and in international patent applications WO 91/02788, WO 96/04394, WO 98/15637, and WO 99/06583. described, each of which is incorporated herein by reference in its entirety.

G. Genetically Modified Cells

In various embodiments, the cell is modified to express a CD33 VHH DARIC contemplated herein, one or more CD33 VHH DARIC components, an anti-CD33 VHH CAR, and/or a fusion protein for use in the treatment of cancer. The cell may be non-genetically modified to express one or more of the polypeptides contemplated herein, or in a particularly preferred embodiment, the cell may be genetically modified to express one or more of the polypeptides contemplated herein. As used herein, the terms "genetically engineered" or "genetically modified" refer to the addition of additional genetic material in the form of DNA or RNA to the total genetic material within a cell. The terms "genetically modified cell", "modified cell", and "reinduced cell" are used interchangeably in certain embodiments.

In certain embodiments, a CD33 VHH DARIC component contemplated herein or an anti-CD33 VHH CAR to improve the efficacy of an immune effector cell is introduced and expressed in an immune effector cell.

An “immune effector cell” is any cell of the immune system that has one or more effector functions (eg, cytotoxic cell death activity, secretion of cytokines, induction of ADCC and/or CDC). Exemplary immune effector cells contemplated herein are T lymphocytes, including but not limited to T cells (CTL; CD8 ⁺ T cells), TILs, and helper T cells (HTL; CD4 ⁺ T cells). . In certain embodiments, the cell comprises an αβ T cell. In certain embodiments, the cell comprises a γδ T cell. In one embodiment, the immune effector cells comprise natural killer (NK) cells. In one embodiment, the immune effector cells comprise natural killer T (NKT) cells. Immune effector cells can be autologous/autogeneic (“self”) or non-autologous (“non-self”, eg, allogeneic, syngeneic, or heterogeneous).

As used herein, “autologous” refers to cells from the same subject. As used herein, “allogenic” refers to a cell of the same species that is genetically different from the cell being compared. As used herein, “syngeneic” refers to a cell from a different subject that is genetically identical to the cell to be compared. As used herein, “xenogeneic” refers to a cell of a different species than the cell being compared. In a preferred embodiment, the cell is a human autoimmune effector cell.

Exemplary immune effector cells suitable for introducing one or more CD33 VHH DARIC components or anti-CD33 VHH CARs contemplated herein include T lymphocytes. The term “T cell” or “T lymphocyte” is intended to include thymocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes. The T cell may be a T helper (Th) cell, for example a T helper 1 (Th1) or T helper 2 (Th2) cell. T cells are helper T cells (HTL; CD4 ⁺ T cells), cytotoxic T cells (CTL; CD8 ⁺ T cells), CD4 ⁺ CD8 ⁺ T cells, CD4 ^- CD8 ^- T cells, or any other subtype of T cells. It can be a set. In certain embodiments, the T cell expresses a T cell receptor. The T cell receptor contains two subunits, the alpha chain and beta chain subunit (αβTCR), or the gamma chain and delta chain subunit (γδTCR), each of which is produced by a recombination event in the genome of the respective T cell. is a unique protein. In certain embodiments, the T cell is an αβTCR T cell (αβ T cell). In certain embodiments, the T cell is a γδ TCR T cell (γδ T cell). Other exemplary T cell populations suitable for use in certain embodiments include untreated T cells and memory T cells.

As will be appreciated by one of ordinary skill in the art, other cells comprising one or more CD33 VHH DARIC components or anti-CD33 VHH CARs contemplated herein may also be used as immune effector cells. In certain embodiments, immune effector cells also include NK cells, NKT cells, neutrophils, and macrophages. Immune effector cells also include progenitor cells of effector cells, which progenitor cells in vivo or can be induced to differentiate into immune effector cells in vitro. Thus, in certain embodiments, the immune effector cells are derived from umbilical cord blood, bone marrow, or mobilized peripheral blood that, upon administration in a subject, differentiate into mature immune effector cells, or which can be induced in vitro to differentiate into mature immune effector cells. Cellular CD34 ⁺ including progenitor cells of immune effector cells such as hematopoietic stem cells (HSCs) contained within the population.

As used herein, the term “CD34 ⁺ cell” refers to a cell that expresses the CD34 protein on its cell surface. As used herein, “CD34” refers to a cell surface glycoprotein (eg, sialomucin protein) that often acts as a cell-cell adhesion factor and is involved in entry of T cells into lymph nodes. The CD34 ⁺ cell population contains hematopoietic stem cells (HSCs), which upon administration to a patient differentiate and contribute to all hematopoietic lineages including T cells, NK cells, NKT cells, neutrophils, and cells of the monocyte/macrophage lineage. .

Methods of making immune effector cells expressing one or more CD33 VHH DARIC components contemplated herein or anti-CD33 VHH CARs are provided in certain embodiments. In one embodiment, the method comprises administering immune effector cells isolated from an individual to one or more nucleic acids and/or vectors, e.g., nucleic acids encoding one or more CD33 VHH DARIC components contemplated herein or anti-CD33 VHH CARs. transfecting or transducing with a lentiviral vector. In one embodiment, the method comprises transfecting or transducing an immune effector cell such that the immune effector cell isolated from the individual expresses one or more CD33 VHH DARIC components contemplated herein and an anti-CD33 VHH CAR. do. In certain embodiments, immune effector cells are isolated from the subject and genetically modified in vitro without further manipulation. These cells can then be re-administered directly into the subject. In a further embodiment, the immune effector cell is first activated, stimulated and proliferated in vitro prior to being genetically modified. In this regard, immune effector cells may be cultured before and/or after being genetically modified.

In certain embodiments, the source of cells is obtained from a subject prior to in vitro manipulation or genetic modification of immune effector cells described herein. In certain embodiments, the modified immune effector cell comprises a T cell.

T cells can be obtained from a number of sources including, but not limited to, peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments, T cells can be treated using any number of techniques known to those of skill in the art, for example, FICOLL?? It can be obtained from blood units taken from a subject using sedimentation, such as separation.

In another embodiment, an isolated or purified T cell population is used. In some embodiments, following isolation of PBMCs, both cytotoxic T lymphocytes and helper T lymphocytes can be sorted into untreated, memory, and effector T cell subpopulations before or after activation, proliferation, and/or genetic modification.

In one embodiment, the isolated or purified population of T cells expresses one or more of the markers including, but not limited to, CD3 ⁺ , CD4 ⁺ , CD8 ⁺ , or combinations thereof.

In certain embodiments, T cells are isolated from a subject and then first activated, stimulated and expanded in vitro prior to being modified to express one or more CD33 VHH DARIC components or anti-CD33 VHH CARs.

In order to obtain a sufficient therapeutic dose of the T cell composition, the T cell is often subjected to one or more stimulation, activation and/or proliferation. In certain embodiments, T cells are generally described, eg, in U.S. Patent Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; and 6,867,041, each of which is incorporated herein by reference in its entirety. In certain embodiments, the T cell is administered for about 6 hours, about 12 hours, about 18 hours, or about 24 hours, optionally before introducing a vector or polynucleotide encoding one or more CD33 VHH DARIC components or anti-CD33 VHH CARs. Activated and proliferated with the engineered antigen receptor contemplated herein.

H. Compositions and Formulations

Compositions contemplated herein include polynucleotides encoding one or more CD33 VHH DARIC components or anti-CD33 VHH CARs, one or more CD33 VHH DARIC components or anti-CD33 VHH CARs, vectors comprising same, genetically modified immune effector cells, bridging factors and the like. Compositions include, but are not limited to, pharmaceutical compositions. "Pharmaceutical composition" refers to a composition formulated as a pharmaceutically acceptable or physiologically acceptable solution for administration to cells or animals alone or in combination with one or more other therapies. It is also noted that, if desired, the composition may be administered in combination with other agents, such as, for example, cytokines, growth factors, hormones, small molecules, chemotherapeutic agents, prodrugs, drugs, antibodies, or various other pharmaceutically active agents. will understand There are virtually no restrictions on other ingredients that may be included in the composition, so long as the additional agent does not adversely affect the ability of the composition to deliver the intended therapy.

The phrase "pharmaceutically acceptable" means, within the scope of sound medical judgment, suitable for use in contact with human and animal tissues without undue toxicity, irritation, allergic reaction, or other problems or complications, and has a reasonable benefit/ Used herein to refer to a compound, substance, composition, and/or dosage form corresponding to a hazard ratio.

The term “pharmaceutically acceptable carrier” refers to a diluent, adjuvant, excipient, or vehicle with which a crosslinking factor, polypeptide, polynucleotide, vector comprising the same, or genetically modified immune effector cell is administered. Exemplary embodiments of pharmaceutical carriers can be sterile liquids such as cell culture media, water, and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. do. Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly as injectable solutions. In certain embodiments suitable pharmaceutical excipients are starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dry skim milk, glycerol, propylene, glycol , water, ethanol, and the like. Except insofar as any conventional media or formulation is incompatible with the active ingredient, its use in therapeutic compositions is contemplated. Supplementary active ingredients may also be incorporated into the compositions.

In one embodiment, a composition comprising a pharmaceutically acceptable carrier is suitable for administration to a subject. In certain embodiments, compositions comprising a carrier are suitable for parenteral administration, eg, intravascular (intravenous or intraarterial), intraperitoneal, or intramuscular administration. In certain embodiments, compositions comprising a pharmaceutically acceptable carrier are suitable for intraventricular, intrathecal, or intrathecal administration. Pharmaceutically acceptable carriers include sterile aqueous solutions, cell culture media, or dispersions. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the bridging factor, polypeptide, polynucleotide, vector comprising the same, or genetically modified immune effector cell, its use in pharmaceutical compositions is contemplated.

In certain embodiments, a composition contemplated herein comprises a genetically modified T cell and a pharmaceutically acceptable carrier. Compositions comprising cell-based compositions contemplated herein may be administered separately by enteral or parenteral administration, or may be administered in combination with other suitable compounds that affect the desired therapeutic goal.

In certain embodiments, compositions contemplated herein include a crosslinking factor and a pharmaceutically acceptable carrier.

A pharmaceutically acceptable carrier should have a sufficiently high purity and sufficiently low toxicity to be suitable for administration to a human subject under treatment. This should further maintain or increase the stability of the composition. Pharmaceutically acceptable carriers can be liquid or solid and are selected with the mode of administration in mind designed to provide the desired bulk, consistency, etc. when combined with the other ingredients of the composition. For example, pharmaceutically acceptable carriers include binders (such as pregelatinized corn starch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose, etc.), fillers (such as lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates, calcium hydrogen phosphate, etc.), lubricants (e.g. magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metal stearate, hydrogenated vegetable oil, corn starch, polyethylene glycol, sodium benzoate, sodium acetate, etc.), a disintegrant (eg, starch, sodium starch glycolate, etc.), or a wetting agent (eg, sodium lauryl sulfate, etc.), but is not limited thereto. Other pharmaceutically acceptable carriers suitable for the compositions contemplated herein include water, salt solutions, alcohols, polyethylene glycol, gelatin, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone, and the like. including, but not limited to.

Such carrier solutions may contain buffers, diluents and other suitable additives. As used herein, the term “buffer” refers to a solution or liquid having a chemical makeup that neutralizes an acid or base without significant change in pH. Examples of buffers contemplated herein include, but are not limited to, Dulbecco's Phosphate Buffered Saline (PBS), Ringer's Solution, 5% Dextrose in Water (D5W), Normal/Physical Saline (0.9% NaCl). .

A pharmaceutically acceptable carrier may be present in an amount sufficient to maintain the pH of the composition at about 7. Alternatively, the composition has a pH in the range of about 6.8 to about 7.4, such as 6.8, 6.9, 7.0, 7.1, 7.2, 7.3 and 7.4. In another embodiment, the composition has a pH of about 7.4.

Compositions contemplated herein may include a non-toxic pharmaceutically acceptable medium. The composition may be a suspension. As used herein, the term “suspension” refers to non-adherent conditions in which cells do not adhere to a solid support. For example, cells maintained as a suspension may be stirred or agitated and not attached to a support such as a culture dish.

In certain embodiments, the compositions contemplated herein are formulated as suspensions, in which case variants?? T cells are dispersed in an acceptable liquid medium or solution (eg, saline or serum-free medium), such as in an intravenous (IV) bag. Acceptable diluents include, but are not limited to, water, PlasmaLyte, Ringer's solution, isotonic sodium chloride (saline) solution, serum-free cell culture media, and media suitable for cryogenic storage, such as Cryostor® media.

In certain embodiments, the pharmaceutically acceptable carrier is substantially free of natural proteins of human or animal origin and is suitable for storing a composition comprising a population of modified T cells. The therapeutic composition is intended to be administered to a human patient and is therefore substantially free of cell culture components such as bovine serum albumin, horse serum, and fetal bovine serum.

In some embodiments, the composition is formulated in a pharmaceutically acceptable cell culture medium. Such compositions are suitable for administration to a human subject. In certain embodiments, the pharmaceutically acceptable cell culture medium is a serum-free medium.

Serum-free media have several advantages over serum-containing media, including simplified and better-defined composition, reduced contamination, elimination of potential sources of infectious agents, and lower cost. In various embodiments, the serum-free medium is animal-free and may optionally be protein-free. Optionally, the medium may contain a biopharmaceutically acceptable recombinant protein. An “animal-free” medium refers to a medium whose components are derived from non-animal sources. Recombinant proteins replace natural animal proteins in animal-free media, and nutrients are obtained from synthetic, plant, or microbial sources. In contrast, a “protein-free” medium is defined as substantially free of protein.

Illustrative examples of serum-free media used in certain compositions include, but are not limited to, QBSF-60 (Quality Biological, Inc.), StemPro-34 (Life Technologies), and X-VIVO 10.

In one embodiment, the composition comprising modified T cells is formulated with PlasmaLyte.

In various embodiments, a composition comprising modified T cells is formulated in a cryopreservation medium. For example, cryopreservation media with cryopreservatives can be used to maintain high cell viability results after thawing. Illustrative examples of cryopreservation media used in certain compositions include, but are not limited to, CryoStor CS10, CryoStor CS5, and CryoStor CS2.

In one embodiment, the composition is formulated as a solution comprising PlasmaLyte A and CryoStor CS10 in a ratio of 50:50.

In certain embodiments, the composition is substantially free of mycoplasma, endotoxin, and microbial contamination. "Substantially free" in the context of endotoxin means that there is less endotoxin per cell dose than FDA allows for biological products, which is equivalent to 5 EU total endotoxin per kg body weight per day. This corresponds to 350 EU per total cell dose for a person with an average body weight of 70 kg. In certain embodiments, a composition contemplated herein comprises from about 0.5 EU/ml to about 5.0 EU/ml, or about 0.5 EU/ml, 1.0 EU/ml, 1.5 EU/ml, 2.0 EU/ml, 2.5 EU/ml, 3.0 EU/ml, 3.5 EU/ml, 4.0 EU/ml, 4.5 EU/ml, or 5.0 EU/ml.

In certain embodiments, formulations of pharmaceutically acceptable carrier solutions are well known to those skilled in the art as well as those skilled in the art to develop appropriate dosing and treatment regimens for use of the particular compositions described herein in a variety of treatment regimens, and appropriate dosing and treatment regimens. These include, for example, enteral and parenteral administration and formulations such as intravascular, intravenous, intraarterial, intraosseous, intraventricular, intracerebral, intracranial, intrathecal, intrathecal, and intramedullary administration and formulations. Included. One of ordinary skill in the art will appreciate that certain embodiments contemplated herein are well known in the art of pharmacy and may include other formulations, such as those described, for example, in Remington: The Science and Practice of Pharmacy , volume I and volume II. 22nd Edition. Edited by Loyd V. Allen Jr. Philadelphia, PA: Pharmaceutical Press; 2012 (incorporated herein by reference in its entirety).

In certain embodiments, the composition comprises an amount of immune effector cells comprising a polynucleotide encoding one or more CD33 VHH DARIC components contemplated herein or an anti-CD33 VHH CAR. In certain embodiments, the composition comprises an amount of immune effector cells expressing one or more CD33 VHH DARIC components contemplated herein or an anti-CD33 VHH CAR. As used herein, the term “amount” means that cells comprising one or more CD33 VHH DARIC components or anti-CD33 VHH CARs, etc. contemplated herein, are beneficial or desirable prophylactic or therapeutic in the presence of a bridging factor. Refers to “an amount effective” or “an effective amount” to achieve a desired result (including clinical results).

A “prophylactically effective amount” is an amount effective to achieve the desired prophylactic result in the presence of a bridging factor, such as cells comprising one or more CD33 VHH DARIC components or anti-CD33 VHH CARs contemplated herein. refers to Because a prophylactic dosage is used in a subject prior to or at an early stage of a disease, a prophylactically effective amount is usually, but not necessarily, a therapeutically effective amount.

A “therapeutically effective amount” means that cells comprising one or more CD33 VHH DARIC components or anti-CD33 VHH CARs contemplated herein “treat” a subject (eg, a patient) in the presence of a bridging factor. amount effective for When a therapeutic amount is indicated, the exact amount of the composition to be administered, cells, bridging factors, etc. can be determined by a doctor in consideration of individual differences in age, weight, tumor size, degree of infection or metastasis, and the condition of the patient (subject). there is.

In general, a pharmaceutical composition comprising immune effector cells as described herein contains 10 ² to 10 ¹⁰ cells per kg body weight, preferably 10 ⁵ to 10 ⁶ cells per kg body weight, including all integer values within these ranges. ) can be administered at a dose of The number of cells will depend on the composition as well as the intended end use of the cell type contained therein. For the uses provided herein, cells are generally in a volume of 1 liter or less, and may be 500 ml or less, even 250 ml or 100 ml or less. Accordingly, the desired density of cells is generally higher than 10 ⁶ cells/ml, generally higher than 10 ⁷ cells/ml, and typically at least 10 ⁸ cells/ml. The number of clinically flexible immune cells can be divided into multiple injections, and the cumulative number is 10 ⁵ , 10 ⁶ , 10 ⁷ , 10 ⁸ , 10 ⁹ , 10 ¹⁰ , 10 ¹¹ , or 10 ¹² cells or more. am. In some embodiments, a lower number of cells (10 ⁶ to 10 ¹¹ per patient) may be administered in the range of 10 ⁶ /kg body weight, particularly since all injected cells will be redirected against a particular target antigen. there is.

If desired, treatment can include mitogens (eg, PHA) or lymphokines, cytokines, and/or chemokines (eg, IFN-γ, IL-2, IL) as described herein to enhance induction of an immune response. -12, TNF-alpha, IL-18, and TNF-beta, GM-CSF, IL-4, IL-13, Flt3-L, RANTES, MIP1α, etc.).

In general, compositions comprising activated and proliferated cells as described herein can be used for the treatment and prevention of diseases occurring in immunocompromised individuals. In particular, the compositions contemplated herein are used for the treatment of cancer. In certain embodiments, the immune effector cells may be administered alone or as a pharmaceutical composition in combination with a carrier, diluent, excipient, and/or other ingredient such as IL-2 or other cytokines or cell populations.

In certain embodiments, the pharmaceutical composition comprises an amount of genetically modified T cells in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients.

In certain embodiments, the pharmaceutical composition comprises an amount of a crosslinking factor in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients.

In certain embodiments, the composition comprises, alone, an effective amount of an immune effector cell comprising one or more CD33 VHH DARIC components contemplated herein or an anti-CD33 VHH CAR, a bridging factor and/or one or more therapeutic agents, such as radiation therapy, including in combination with chemotherapy, graft, immunotherapy, hormonal therapy, photodynamic therapy, and the like. The composition may also be administered in combination with an antibiotic. Such therapeutic agents are acceptable in the art as standard of care for certain conditions as described herein, such as certain cancers. Exemplary therapeutic agents contemplated include cytokines, growth factors, steroids, NSAIDs, DMARDs, anti-inflammatory agents, chemotherapeutic agents, radiotherapeutic agents, therapeutic antibodies, or other active agents and adjuvants.

In certain embodiments, a composition comprising an effective amount of immune effector cells comprising a polynucleotide encoding one or more CD33 VHH DARIC components contemplated herein or an anti-CD33 VHH CAR is administered to a subject and comprises an effective amount of a bridging factor. The composition is administered to the subject prior to, during, concurrently with, or after administration of the cell composition, and optionally repeatedly administered to the subject.

In certain embodiments, a composition comprising immune effector cells comprising a polynucleotide encoding one or more CD33 VHH DARIC components or anti-CD33 VHH CARs contemplated herein comprises any number of anti-inflammatory agents, chemotherapeutic agents, or therapeutic agents, etc. may be administered together.

I. METHODS OF TREATMENT

Immune effector cells that have been modified to express one or more CD33 VHH DARIC components or polynucleotides encoding an anti-CD33 VHH CAR contemplated herein prevent, treat, and ameliorate an immune disorder (eg, cancer), or at least one associated with it. An improved adoptive immunotherapy for use in preventing, treating, or alleviating the symptoms of

Immune effector cells comprising a CD33 DARIC signaling component, a CD33 VHH DARIC binding component, or an anti-CD33 VHH CAR prevent, treat, and ameliorate an immune disorder (eg, cancer), or prevent at least one symptom associated therewith; An improved adoptive immunotherapy for use in treatment and palliation is provided.

In certain embodiments, immune effector cells modified to express CD33 VHH DARIC express target antigen while reducing the risk of target antigen correct, target cell cytotoxicity inaccuracy (recognizing target antigen in normal non-target cells). To provide improved adoptive immunotherapy to fine-tune the safety and efficacy of cytotoxic responses against target cells (eg, tumor cells).

In certain embodiments, a method of preventing, treating, or ameliorating at least one symptom of cancer comprises administering to a subject an effective amount of a modified immune effector cell or T cell comprising one or more components of a CD33 VHH DARIC or anti-CD33 VHH CAR. and reinducing these cells against the target cells by administering to the Genetically modified cells are a more effective and safer cellular immunotherapy by transducing chemically regulatable immunostimulatory signals.

In certain embodiments, one or more immune effector cells (eg, T cells) are modified to express both a CD33 VHH DARIC binding component and a CD33 VHH DARIC signaling component. In this case, the modified cells are administered to a subject in need thereof and administered to the home of the target cells via interaction of the CD33 VHH DARIC binding component expressed on the immune effector cells with CD33 expressed on the target cells. The bridging factor is administered to a subject before the modified cells are administered to the subject, at about the same time as the modified cells, or after the modified cells are administered to the subject. In the presence of a bridging factor, a ternary complex is formed between the CD33 VHH DARIC binding component, the bridging factor, and the CD33 DARIC signaling component. Upon formation of the ternary complex, CD33 VHH DARIC transmits an immunostimulatory signal to immune effector cells, which ultimately elicit a cytotoxic response from the immune effector cells against target cells.

In certain embodiments, one or more immune effector cells (eg, T cells) are modified to express a CD33 DARIC signaling component. In this case, the modified cells are administered to a subject in need thereof. The CD33 VHH DARIC binding component can be administered to the subject before the modified cells are administered to the subject, at about the same time as the modified cells, or after the modified cells are administered to the subject. In addition, the CD33 VHH DARIC binding component is administered to the subject as a preformed complex with a crosslinking factor; administered concurrently with the crosslinking factor, but as a separate composition; It may be administered at a different time than the bridging factor. The CD33 VHH binding component binds to CD33 expressed on target cells in the presence or absence of a bridging factor. In the presence of a bridging factor, a ternary complex is formed between the CD33 VHH DARIC binding component, the bridging factor, and the CD33 DARIC signaling component. Upon formation of the ternary complex, CD33 VHH DARIC transmits an immunostimulatory signal to immune effector cells, which ultimately elicit a cytotoxic response from the immune effector cells against target cells.

In certain embodiments, one or more immune effector cells (eg, T cells) are modified to express a CD33 DARIC signaling component. In this case, the modified cells are administered to a subject in need thereof. The CD33 VHH DARIC binding component can be administered to the subject before the modified cells are administered to the subject, at about the same time as the modified cells, or after the modified cells are administered to the subject. In addition, the CD33 VHH DARIC binding component is administered to the subject as a preformed complex with a crosslinking factor; administered concurrently with the crosslinking factor, but as a separate composition; It may be administered at a different time than the bridging factor. The CD33 binding component binds to a target antigen expressed on target cells in the presence or absence of a bridging factor. In the presence of a bridging factor, a ternary complex is formed between the CD33 VHH DARIC binding component, the bridging factor, and the CD33 DARIC signaling component. Upon formation of the ternary complex, CD33 VHH DARIC transmits an immunostimulatory signal to immune effector cells, which ultimately elicit a cytotoxic response from the immune effector cells against target cells. In certain embodiments, CD33 VHH DARIC activation can be induced when remission or regression is incomplete and the condition recurs or becomes refractory to treatment.

In certain preferred embodiments, the specificity of the primary T cell is re-induced for a tumor or cancer cell expressing CD33 by genetically modifying the T cell (eg primary T cell) with one or more CD33 VHH DARIC components.

In certain preferred embodiments, the specificity of the primary T cell is achieved by genetically modifying the T cell (eg primary T cell) with an engineered antigen receptor directed against a target antigen and one or more CD33 VHH DARIC components, thereby resulting in a tumor expressing CD33. or against cancer cells.

In certain embodiments, the modified immune effector cells contemplated herein are used in the treatment of solid tumors or cancers.

In certain embodiments, the modified immune effector cells contemplated herein are used in the treatment of solid tumors or cancers, including but not limited to: adrenal cancer, adrenocortical carcinoma, anal cancer, appendix cancer, astrocytoma, Atypical malformation/rhabdomyocarcinoma, basal cell carcinoma, cholangiocarcinoma, bladder cancer, bone cancer, brain/CNS cancer, breast cancer, bronchial tumor, heart tumor, cervical cancer, cholangiocarcinoma, chondrosarcoma, chordoma, colon cancer, colorectal cancer, craniopharyngoma, duct Intraepithelial carcinoma (DCIS), endometrial cancer, ependymal celloma, esophageal cancer, sciatic neuroblastoma, Ewing sarcoma, extracranial germ cell tumor, extragonadal germ cell tumor, eye cancer, fallopian tube cancer, fibrous histosarcoma, fibrosarcoma, gallbladder cancer, Gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), germ cell tumor, glioma, glioblastoma, head and neck cancer, hemangioblastoma, hepatocellular carcinoma, hypopharyngeal cancer, intraocular melanoma, Kaposi's sarcoma, renal cancer, laryngeal cancer, smooth muscle sarcoma, labial cancer, liposarcoma, liver cancer, lung cancer, non-small cell lung cancer, lung carcinoma, malignant mesothelioma, medullary carcinoma, medulloblastoma, meningioma, melanoma, melanoma, Merkel cell carcinoma, mesangial duct carcinoma, oral cancer, myxosarcoma, myelodysplastic syndrome, Myeloproliferative neoplasm, nasal and sinus cancer, nasopharyngeal cancer, neuroblastoma, oligodendroglioma, oral cancer, oral cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, pancreatic islet cell tumor, papillary carcinoma, paraganglioma, parathyroid cancer, Penile cancer, pharyngeal cancer, pheochromocytoma, pineal tumor, pituitary tumor, pleuropulmonary blastoma, primary peritoneal cancer, prostate cancer, rectal cancer, retinoblastoma, renal cell carcinoma, renal pelvic and ureter cancer, rhabdomyosarcoma, salivary gland cancer, sebaceous gland carcinoma, Skin cancer, soft tissue sarcoma, squamous cell carcinoma, small cell lung cancer, small intestine cancer, stomach cancer, sweat gland carcinoma, synovoma, testicular cancer, throat cancer, thymus cancer, thyroid cancer, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, vascular cancer, vulvar cancer, and bill Rum's tumor.

In certain embodiments, the modified immune effector cells contemplated herein are non-small cell lung carcinoma, head and neck squamous cell carcinoma, colorectal cancer, pancreatic cancer, breast cancer, thyroid cancer, bladder cancer, cervical cancer, esophageal cancer, ovarian cancer, gastric cancer, endometrial cancer, It is used in the treatment of solid tumors or cancers, including but not limited to gliomas, gliomas, and oligodendrogliomas.

In certain embodiments, the modified immune effector cells contemplated herein are used in the treatment of solid tumors or cancers, including but not limited to non-small cell lung cancer, metastatic colorectal cancer, neuroblastoma, head and neck cancer, pancreatic cancer, and breast cancer. .

In certain embodiments, the modified immune effector cells contemplated herein are used in the treatment of neuroblastoma.

In certain embodiments, the modified immune effector cells contemplated herein are used in the treatment of liquid cancer or hematologic cancer.

In certain embodiments, the modified immune effector cells contemplated herein are used in the treatment of B cell malignancies including but not limited to leukemia, lymphoma, and multiple myeloma.

In certain embodiments, the modified immune effector cells contemplated herein are used in the treatment of liquid cancer, including but not limited to: leukemia, lymphoma, and multiple myeloma: acute lymphocytic leukemia (ALL), acute myeloid Leukemia (AML), myeloblastic leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia, erythroleukemia, blastic leukemia (HCL), chronic lymphocytic leukemia (CLL), and chronic myelogenous leukemia (CML), Chronic myelomonocytic leukemia (CMML) and polycythemia vera, Hodgkin's lymphoma, nodular lymphocyte-predominant Hodgkin's lymphoma, Burkitt's lymphoma, small lymphocytic lymphoma (SLL), diffuse large B-cell lymphoma, follicular lymphoma, immunoblastoma Large cell lymphoma, precursor B-lymphoblastic lymphoma, mantle cell lymphoma, marginal zone lymphoma, mycosis fungoides, anaplastic large cell lymphoma, Sezary syndrome, precursor T-lymphoblastic lymphoma, multiple myeloma, overt multiple myeloma, subacute multiple Myeloma, plasma cell leukemia, nonsecretory myeloma, IgD myeloma, osteosclerotic myeloma, bone solitary plasmacytoma, and extramedullary plasmacytoma.

In certain embodiments, the modified immune effector cells contemplated herein are used in the treatment of acute myeloid leukemia (AML).

Preferred cells for use in the methods contemplated herein include autologous/autogenic (“self”) cells, preferably hematopoietic cells, more preferably T cells, and more preferably immune effector cells. .

In certain embodiments, the method comprises administering to a patient in need thereof a therapeutically effective amount of a modified immune effector cell expressing one or more CD33 VHH DARIC components, and also administering to the subject a bridging factor. In certain embodiments, the cells are used to treat a patient at risk of developing an immune disorder. Accordingly, certain embodiments include the treatment or prevention or amelioration of at least one symptom of an immune disorder (eg, cancer), wherein the treatment or prophylaxis or amelioration comprises a therapeutically effective amount and crosslinking of the modified immune effector cells contemplated herein. administering the factor to a subject in need thereof.

In certain embodiments, the method comprises administering to a patient in need thereof a therapeutically effective amount of a modified immune effector cell expressing an anti-CD33 VHH CAR. In certain embodiments, the cells are used to treat a patient at risk of developing an immune disorder. Accordingly, certain embodiments include the treatment or prevention or amelioration of at least one symptom of an immune disorder (eg, cancer), wherein the treatment or prophylaxis or amelioration comprises a therapeutically effective amount and crosslinking of the modified immune effector cells contemplated herein. administering the factor to a subject in need thereof.

In certain embodiments, the method comprises administering to a patient in need thereof a therapeutically effective amount of a modified immune effector cell expressing a CD33 DARIC signaling component or a composition comprising the same, and also administering a CD33 VHH DARIC binding component and a crosslinking factor. and optionally wherein the CD33 VHH DARIC binding component is bound to a crosslinking factor prior to administration to the subject. In certain embodiments, the cells are used to treat a patient at risk of developing an immune disorder. Accordingly, certain embodiments include the treatment or prevention or amelioration of at least one symptom of an immune disorder (eg, cancer), wherein the treatment or prevention or amelioration comprises treatment of a modified immune effector cell expressing a CD33 DARIC signaling component. administering an effective amount to a subject in need thereof, and optionally administering an engineered antigen receptor or another DARC binding component, a CD33 VHH DARIC binding component, and a crosslinking factor.

The dosage and frequency of modified immune effector cells, CD33 DARIC VHH binding components, and/or bridging factors are determined by factors such as the patient's condition and the type and severity of the patient's disease, although appropriate dosages and dosing schedules are It can be determined by clinical trials.

In one exemplary embodiment, the effective amount of modified immune effector cells provided to the subject is at least 2 x 10 ⁶ cells/kg body weight, at least 3 x 10 ⁶ cells/kg, at least 4 x 10 ⁶ cells/kg, at least 5 x 10 ⁶ cells/kg, at least 6 x 10 ⁶ cells/kg, at least 7 x 10 ⁶ cells/kg, at least 8 ^x 10 ⁶ cells/kg, at least 9 x 10 ⁶ cells/kg, or at least 10 x 10 ⁶ cells/kg or more, including all intervening cell doses.

In another exemplary embodiment, the effective amount of modified immune effector cells provided to the subject is about 2 x 10 ⁶ cells/kg body weight, about 3 x 10 ⁶ cells/kg, about 4 x 10 ⁶ cells/kg, about 5 x 10 ⁶ cells/kg, about 6 x 10 ⁶ cells/kg, about 7 x 10 ⁶ cells/kg, about 8 ^x 10 ⁶ cells/kg, about 9 x 10 ⁶ cells/kg, or about 10 x 10 ⁶ cells/kg or more, including all intervening cell doses.

In another exemplary embodiment, the effective amount of modified immune effector cells provided to the subject is from about 2 x 10 ⁶ cells/kg body weight to about 10 x 10 ⁶ cells/kg, from about 3 x 10 ⁶ cells/kg to about 10 x 10 ⁶ cells/kg, about 4 x 10 ⁶ cells/kg to about 10 x 10 ⁶ cells/kg, about 5 x 10 ⁶ cells/kg to about 10 x 10 ⁶ cells/kg, 2 x 10 ⁶ cells /kg to about 6 x 10 ⁶ cells/kg, 2 x 10 ⁶ cells/kg to about 7 x 10 ⁶ cells/kg, 2 x 10 ⁶ cells/kg to about 8 x 10 ⁶ cells/kg, 3 x 10 ⁶ cells/kg to about 6×10 ⁶ cells/kg, 3×10 ⁶ cells/kg to about 7×10 ⁶ cells/kg, 3×10 ⁶ cells/kg to about 8×10 ⁶ cells/kg, 4×10 ⁶ cells/kg to about 6 x 10 ⁶ cells/kg, 4 x 10 ⁶ cells/kg to about 7 x 10 ⁶ cells/kg, 4 x 10 ⁶ cells/kg to about 8 x 10 ⁶ cells/kg, 5 x 10 ⁶ cells/kg to about 6×10 ⁶ cells/kg, 5×10 ⁶ cells/kg to about 7×10 ⁶ cells/kg, 5×10 ⁶ cells/kg to about 8×10 ⁶ cells/kg, or 6×10 ⁶ cells/kg to about 8×10 ⁶ cells/kg, including all intervening cell doses.

One of ordinary skill in the art will recognize that in certain embodiments, multiple administrations of contemplated compositions may be required to effect the desired therapy. For example, the composition may be administered in a period of 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, 2 years, 5 years, 10 years, or more. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more. The modified immune effector cells, the CD33 VHH DARIC component, and the bridging factor are administered in the same or different compositions; administered simultaneously in more than one composition; The two or more compositions may be administered at different times. The modified immune effector cells, the CD33 VHH DARIC component, and the bridging factor may be administered by the same route of administration or may be administered via different routes.

In certain embodiments, the activated T cells are administered to the subject and then blood is drawn back (or apheresis is performed), activated T cells therefrom, and the activated and proliferated T cells and expanded T cells. It may be desirable to re-inject the patient. This process can be performed several times every few weeks. In certain embodiments, T cells can be activated from 10 cc to 400 cc of blood draw. In certain embodiments, the T cells are 20 cc, 30 cc, 40 cc, 50 cc, 60 cc, 70 cc, 80 cc, 90 cc, 100 cc, 150 cc, 200 cc, 250 cc, 300 cc, 350 cc, or from 400cc or more blood draw. Without wishing to be bound by theory, using these multiple blood draw/multiple reinfusion protocols may serve to select specific T cell populations.

In one embodiment, a method of treating a subject diagnosed with cancer comprises removing immune effector cells from the subject, introducing into the cell one or more vectors encoding one or more CD33 VHH DARIC components, and generating a population of modified immune effector cells. producing, and administering the population of modified immune effector cells to the same subject. In a preferred embodiment, the immune effector cells comprise T cells.

In one embodiment, a method of treating a subject diagnosed with cancer comprises removing immune effector cells from the subject, introducing one or more vectors encoding an anti-CD33 VHH CAR into the cell and producing a population of modified immune effector cells. and administering the population of modified immune effector cells to the same subject. In a preferred embodiment, the immune effector cells comprise T cells.

Contemplated methods of administering a cell composition in certain embodiments include reintroduction of modified immune effector cells ex vivo, or reintroduction of modified progenitor cells of immune effector cells that, when introduced into a subject, differentiate into mature immune effector cells. any method effective to induce One method involves introducing into a cell an engineered antigen receptor and one or more vectors encoding one or more CD33 VHH DARIC components or anti-CD33 VHH CAR, and injecting the transduced cells back into the subject ex vivo peripheral blood T cells transforming it.

All publications, patent applications, and issued patents cited herein are incorporated herein by reference as if each individual publication, patent application, or issued patent were specifically and individually indicated to be incorporated by reference.

While the foregoing embodiments have been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be appreciated by those skilled in the art in light of the teachings contemplated herein that certain changes and modifications may be made without departing from the spirit or scope of the appended claims. It will be readily apparent. The following examples are provided by way of illustration only and are not intended to be limiting. Those skilled in the art will readily recognize various non-critical parameters that may be altered or modified in the particular implementation to obtain essentially similar results.

Example

Example 1

CD33 VHH DARIC T cells exhibit antitumor responses

Anti-CD33 VHH DARIC binding and signaling components were designed, constructed, and validated. A CD33 specific DARIC lentiviral vector was constructed comprising a MNDU3 promoter operably linked to a polynucleotide encoding the following: a DARIC signal transduction component (CD8α-signal peptide, FRB variant (T82L), CD8α transmembrane domain, intracellular 4-1BB costimulatory domain, and CD3ζ zeta signaling domain); P2A sequence; and a CD4-derived transmembrane domain comprising a DARIC binding component (Igκ-signal peptide, a CD33 specific VHH binding domain (camelliid or humanized), a G4S linker, a FKBP12 domain, and a truncated intracellular domain ( FIG. 1B ). See, for example, SEQ ID NOs: 32-41. T cells transduced with anti-CD33 DARIC lentiviral vectors express the membrane binding polypeptides shown in Figure 1 A. Anti-CD33 scFv CAR or DARIC designs were used as controls. .

T cells from three donors were each transduced with LVV encoding different CD33 specific VHH DARICs, anti-CD33 scFv DARICs, or anti-CD33 scFv CARs and proliferated for 10 days. T cells transduced with untransduced T cells, anti-CD33 scFv control constructs or anti-CD33 VHH DARIC T cells were stained with recombinant CD33-Fc reagent. Only control CAR and DARIC T cells were positively stained with CD33-Fc staining (Fig. 2a, bottom panel, Fig. 2b). However, when analyzed with a monoclonal antibody specific for the VHH domain, most anti-CD33 VHH DARIC T cells, but not control CAR or DARIC T cells, stained positively (Fig. 2a, top panel). Both control CAR and DARIC T cells and anti-CD33 VHH DARIC T cells had similar T cell phenotypes, as determined in part by CD62L and CD45RA staining ( FIGS. 3A and 3B ).

Non-transduced T cells, T cells transduced with anti-CD33 scFv control construct, or T cells transduced with anti-CD33 VHH DARIC T cells were mixed 1: 1 with CD33+ THP-1 cells in the presence or absence of AP21967. Co-cultured for 24 h at an E:T ratio of 1. Anti-CD33 scFv CAR control cells showed robust cytokine production with or without rapalogs. Anti-CD33 scFv DARIC T cells and anti-CD33 VHH DARIC T cells showed a strong cytokine response only when incubated with THP-1 cells in the presence of AP21967 ( FIGS. 4A and 4B ). Minimal cytokine production was detected in the non-transduced control group.

In addition, the specificity of VHH9 and VHH10 DARIC was evaluated for full-length CD33 (CD33M) as well as for splice variants expressing shorter truncated CD33 (CD33m). Human 293T cells were electroporated with mRNA encoding either full-length CD33M or the splice variant CD33m ( FIG. 4C ). DARIC T cells were co-cultured with modified 293T cells in the presence or absence of AP21967 at an E:T ratio of 1:1 for 24 h and assessed for activation as measured by cytokine secretion ( FIG. 4C ). VHH9 DARIC T cells displayed a strong cytokine response in response to CD33M or CD33m 293T cells, whereas VHH10 DARIC T cells were activated only in the presence of CD33M.

Example 2

CD33 VHH DARIC T cells respond specifically to CD33 antigen

Anti-CD33 VHH DARIC T cells were generated as described in Example 1. T cells from three donors were each transduced with LVV encoding a different anti-CD33 specific VHH DARIC and grown for 10 days. Controls included untransduced (UTD) T cells and CD33 CAR transduced T cells. The AML cell line MV4-11 normally expresses CD33. MV4-11 cells were engineered to knock out the CD33 gene (CD33-KO cells). The resulting CD33-KO cell line lacked CD33 expression on the cell surface. Figure 5a. Anti-CD33 VHH DARIC T cells were co-cultured with MV4-11 cells or CD33-KO cells in the presence or absence of dimerization drug at an E:T ratio of 1:1 for 24 hours. Anti-CD33 VHH DARIC T cells produced cytokines in the presence of MV4-11 target cells, but not in the presence of CD33-KO cells. Figure 5b.

The CD33-KO cell line was modified to express the CD33m splice variant (CD33-KO-C2). MV4-11 cells and CD33-KO-C2 cells were co-cultured with UTD cells, anti-CD33 CAR T cells, or anti-CD33 VHH DARIC T cells in the presence or absence of dimerization drug, and cytokine production was inhibited after 24 h. analyzed. Anti-CD33 VHH9 DARIC recognized both normal CD33 and CD33m splice variants and produced cytokines when co-cultured with MV4-11 cells or CD33-KO-C2 cells ( FIG. 5C ). Anti-CD33 CAR T cells or anti-CD33VHH2 DARIC control T cells were only active against MV4-11 target cells.

Example 3

CD33 VHH DARIC T cells are not inhibited by soluble CD33 protein

Anti-CD33 VHH DARIC T cells were generated as described in Example 1. T cells from three donors were each transduced with LVV encoding a different anti-CD33 specific VHH DARIC and grown for 10 days. Anti-CD33 VHH DARIC T cells were co-cultured with CD33 ⁺ THP-1 cells in the presence or absence of rapamycin at an E:T ratio of 1:1 for 24 hours. Various amounts of recombinant CD33-Fc protein were added during the co-culture period. Anti-CD33 VHH DARIC T cells displayed a potent cytokine response in the presence of rapamycin in the presence and absence of recombinant soluble CD33 protein. Fig. 6.

Example 4

CD33 VHH DARIC T cells respond to low levels of CD33 antigen

Anti-CD33 VHH DARIC T cells were generated as described in Example 1. T cells from three donors were each transduced with LVV encoding a different anti-CD33 specific VHH DARIC and grown for 10 days. Anti-CD33 VHH DARIC T cells were co-cultured with CD33 ^neg 293T cells transfected with AP21967 dimerization agent and different amounts of mRNA encoding CD33. After 24 hours, the supernatant was collected and analyzed for cytokine production. Anti-CD33 VHH DARIC T cells showed a dose-dependent increase in IFNγ production even at very low mRNA concentrations after co-culture with CD33-transfected target cells ( FIG. 7 ).

Example 5

CD33 VHH DARIC T cells regulate tumor growth in vivo

Anti-CD33 VHH DARIC T cells were generated as described in Example 1. HL60 AML tumor cells expressing a luciferase reporter were inoculated into immunodeficient NSG mice to establish CD33 expressing tumors in these mice. Tumor growth was monitored by luminescence. After 10 days, mice were co-administered with 10×10 ⁶ anti-CD33 VHH DARIC T cells with vehicle or rapamycin. The control group included mice administered with rapamycin alone or untransduced (UTD) T cells. Tumor growth was similar in both treatment and control groups. 8A Mice treated with anti-CD33 VHH DARIC T cells and rapamycin showed an increase in tumor control compared to mice treated with UTD T cells and rapamycin. Figure 8b.

Generally, in the following claims, the terminology used is not to be construed as limiting the claims to the specification and specific embodiments disclosed in the claims, but to all possible implementations of which such claims are entitled, along with the full scope of equivalents to which they are granted. It should be construed as including examples. Accordingly, the claims are not limited by the present disclosure.

SEQUENCE LISTING <110> bluebird bio, Inc. Inhibrx, Inc. Jarjour, Jordan Pogson, Mark Leung, Wai-Hang Jones, Kyle Crago, William Sanabria, Angelica Hollands, Andrew Gano, Jacob Ma, Milton Timmer, John C. Eckelman, Brendan P. <120> CD33 TARGETED IMMUNOTHERAPIES <130> BLUE-119.PC <150> US 62/898,392 <151> 2019-09-10 <150> US 62/845,304 <151> 2019-05-08 <160> 119 <170> PatentIn version 3.5 <210> 1 <211> 364 <212> PRT <213> Homo sapiens <400> 1 Met Pro Leu Leu Leu Leu Leu Pro Leu Leu Trp Ala Gly Ala Leu Ala 1 5 10 15 Met Asp Pro Asn Phe Trp Leu Gln Val Gln Glu Ser Val Thr Val Gln 20 25 30 Glu Gly Leu Cys Val Leu Val Pro Cys Thr Phe Phe His Pro Ile Pro 35 40 45 Tyr Tyr Asp Lys Asn Ser Pro Val His Gly Tyr Trp Phe Arg Glu Gly 50 55 60 Ala Ile Ile Ser Arg Asp Ser Pro Val Ala Thr Asn Lys Leu Asp Gln 65 70 75 80 Glu Val Gln Glu Glu Thr Gln Gly Arg Phe Arg Leu Leu Gly Asp Pro 85 90 95 Ser Arg Asn Asn Cys Ser Leu Ser Ile Val Asp Ala Arg Arg Arg Asp 100 105 110 Asn Gly Ser Tyr Phe Phe Arg Met Glu Arg Gly Ser Thr Lys Tyr Ser 115 120 125 Tyr Lys Ser Pro Gln Leu Ser Val His Val Thr Asp Leu Thr His Arg 130 135 140 Pro Lys Ile Leu Ile Pro Gly Thr Leu Glu Pro Gly His Ser Lys Asn 145 150 155 160 Leu Thr Cys Ser Val Ser Trp Ala Cys Glu Gln Gly Thr Pro Pro Ile 165 170 175 Phe Ser Trp Leu Ser Ala Ala Pro Thr Ser Leu Gly Pro Arg Thr Thr 180 185 190 His Ser Ser Val Leu Ile Ile Thr Pro Arg Pro Gln Asp His Gly Thr 195 200 205 Asn Leu Thr Cys Gln Val Lys Phe Ala Gly Ala Gly Val Thr Thr Glu 210 215 220 Arg Thr Ile Gln Leu Asn Val Thr Tyr Val Pro Gln Asn Pro Thr Thr 225 230 235 240 Gly Ile Phe Pro Gly Asp Gly Ser Gly Lys Gln Glu Thr Arg Ala Gly 245 250 255 Val Val His Gly Ala Ile Gly Gly Ala Gly Val Thr Ala Leu Leu Ala 260 265 270 Leu Cys Leu Cys Leu Ile Phe Phe Ile Val Lys Thr His Arg Arg Lys 275 280 285 Ala Ala Arg Thr Ala Val Gly Arg Asn Asp Thr His Pro Thr Thr Gly 290 295 300 Ser Ala Ser Pro Lys His Gln Lys Lys Ser Lys Leu His Gly Pro Thr 305 310 315 320 Glu Thr Ser Ser Cys Ser Gly Ala Ala Pro Thr Val Glu Met Asp Glu 325 330 335 Glu Leu His Tyr Ala Ser Leu Asn Phe His Gly Met Asn Pro Ser Lys 340 345 350 Asp Thr Ser Thr Glu Tyr Ser Glu Val Arg Thr Gln 355 360 <210> 2 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> Made in lab - anti-CD33 VHH sequence <400> 2 Gln Val Thr Leu Arg Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Lys Gly Ser Gly Arg Ala Ile Asn Thr Tyr 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Ala Ile Ser Trp Asn Gly Gly His Thr Arg Tyr Ala Asp Ser Val 50 55 60 Gln Gly Arg Phe Ala Ile Ser Arg Asp Asn Ala Asp Asn Thr Met Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr His Cys 85 90 95 Ala Ala Tyr Ser Asp Tyr His Arg Ile Ala Thr Met Glu Ala Asp Ala 100 105 110 Asp Ser Trp Gly Gly Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 3 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Made in lab - anti-CD33 VHH sequence <400> 3 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Glu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Gly Tyr 20 25 30 Ile Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val 35 40 45 Ala Arg Ile Ser Gly Asn Asn Leu Ser Thr Glu Tyr Ala Glu Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Glu Tyr Asp Tyr Ser Ser Gly Asp Phe Val Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Lys Pro 115 120 <210> 4 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Made in lab - anti-CD33 VHH sequence <400> 4 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Glu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Gly Phe Ser Ala Ser 20 25 30 Leu Met Ser Trp His Arg Gln Ala Pro Gly Lys Gln Arg Asp Leu Val 35 40 45 Ala Ser Ile Thr Arg Asp Gly Arg Ala Asn Tyr Val Glu Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys His 85 90 95 Ala Tyr Ser Phe Asp Tyr Pro Ile Arg Ser Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Lys Pro 115 <210> 5 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Made in lab - anti-CD33 VHH sequence <400> 5 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Glu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Trp Val 35 40 45 Ala Ala Ile Thr Thr Ser Gly Asp Thr Thr Tyr Tyr Ala Glu Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala His Arg Gly Gly Gly Val Ile Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Lys Pro 115 <210> 6 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Made in lab - anti-CD33 VHH sequence <400> 6 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Glu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Phe Ser Ile Ser 20 25 30 Ile Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val 35 40 45 Ala Ser Thr Thr Ser Ser Gly Thr Thr Asn Tyr Val Glu Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys His 85 90 95 Ala Tyr Ile Ala Thr Thr Thr Asp Arg Gly Tyr Arg Gly Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Lys Pro 115 120 <210> 7 <211> 124 <212> PRT <213> Artificial Sequence <220> <223> Made in lab - anti-CD33 VHH sequence <400> 7 Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Ser Ser Ser Tyr Asn 20 25 30 Val Val Gly Trp Tyr Arg Gln Leu Ser Gly Asn Glu Arg Gly Gly Arg 35 40 45 Thr Met Val Ala Gln Ile Asn Ala Tyr Gly Asp Thr Asn Tyr Ala Asn 50 55 60 Ala Val Val Gly Arg Phe Thr Ile Ser Arg Asp Asp Ala Lys Asn Thr 65 70 75 80 Val Tyr Leu His Met Ser Asn Leu Lys Pro Glu Asp Thr Gly Val Tyr 85 90 95 Tyr Cys Asn Gly Gln Arg Met Leu Glu Asn Tyr Thr Tyr Arg Asp Gln 100 105 110 Ser Trp Gly Gln Gly Thr Gln Val Thr Val Lys Pro 115 120 <210> 8 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> Made in lab - anti-CD33 VHH sequence <400> 8 Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Thr Leu Ser Cys Ala Ala Ser Arg Ser Ser Gly Ile Asp Val 20 25 30 Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val Ala 35 40 45 Glu Ile Ser Gly Val Gly Asp Thr Asn Tyr Ala Ala Ser Leu Ala Asp 50 55 60 Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln 65 70 75 80 Met Lys Asn Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn Ala 85 90 95 His Ser Phe Leu Asp Leu Val Gly Ala Trp Gly Gln Gly Thr Gln Val 100 105 110 Thr Val Lys Pro 115 <210> 9 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Made in lab - anti-CD33 VHH sequence <400> 9 Glu Val Gln Leu Val Gln Ser Gly Gly Gly Ser Val Gln Val Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Leu Asn Ile Asp 20 25 30 His Ile Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val 35 40 45 Gly Val Ile Ser Ser Gly Ala Gly Pro Asn Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Asn Cys Asn 85 90 95 Ala Trp Ile Asp Tyr Gly Ser Gly Leu Pro Gln Asn Tyr Trp Gly Gln 100 105 110 Gly Thr Gln Val Thr Val Lys Pro 115 120 <210> 10 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> Made in lab - anti-CD33 VHH sequence <400> 10 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Glu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Arg Ser Ser Gly Ile Asp Val 20 25 30 Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val Ala 35 40 45 Glu Ile Ser Gly Val Gly Asp Thr Asn Tyr Ala Ala Ser Leu Ala Asp 50 55 60 Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln 65 70 75 80 Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Asn Ala 85 90 95 His Ser Phe Leu Asp Leu Val Gly Ala Trp Gly Gin Gly Thr Leu Val 100 105 110 Thr Val Lys Pro 115 <210> 11 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Made in lab - anti-CD33 VHH sequence <400> 11 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Glu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Leu Asn Ile Asp 20 25 30 His Ile Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val 35 40 45 Gly Val Ile Ser Ser Gly Ala Gly Pro Asn Tyr Ala Glu Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Asn 85 90 95 Ala Trp Ile Asp Tyr Gly Ser Gly Leu Pro Gln Asn Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Lys Pro 115 120 <210> 12 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> Made in lab - anti-CD33 VHH sequence <220> <221> MOD_RES <222> (124)..(125) <223> Xaa is any amino acid or absent <400> 12 Gln Val Thr Leu Arg Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Lys Gly Ser Gly Arg Ala Ile Asn Thr Tyr 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Ala Ile Ser Trp Asn Gly Gly His Thr Arg Tyr Ala Asp Ser Val 50 55 60 Gln Gly Arg Phe Ala Ile Ser Arg Asp Asn Ala Asp Asn Thr Met Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr His Cys 85 90 95 Ala Ala Tyr Ser Asp Tyr His Arg Ile Ala Thr Met Glu Ala Asp Ala 100 105 110 Asp Ser Trp Gly Gin Gly Thr Gln Val Thr Val Xaa Xaa 115 120 125 <210> 13 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Made in lab - anti-CD33 VHH sequence <220> <221> MOD_RES <222> (119)..(120) <223> Xaa is any amino acid or absent <400> 13 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Glu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Gly Tyr 20 25 30 Ile Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val 35 40 45 Ala Arg Ile Ser Gly Asn Asn Leu Ser Thr Glu Tyr Ala Glu Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Glu Tyr Asp Tyr Ser Ser Gly Asp Phe Val Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Xaa Xaa 115 120 <210> 14 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Made in lab - anti-CD33 VHH sequence <220> <221> MOD_RES <222> (117)..(118) <223> Xaa is any amino acid or absent <400> 14 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Glu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Gly Phe Ser Ala Ser 20 25 30 Leu Met Ser Trp His Arg Gln Ala Pro Gly Lys Gln Arg Asp Leu Val 35 40 45 Ala Ser Ile Thr Arg Asp Gly Arg Ala Asn Tyr Val Glu Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys His 85 90 95 Ala Tyr Ser Phe Asp Tyr Pro Ile Arg Ser Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Xaa Xaa 115 <210> 15 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Made in lab - anti-CD33 VHH sequence <220> <221> MOD_RES <222> (117)..(118) <223> Xaa is any amino acid or absent <400> 15 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Glu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Trp Val 35 40 45 Ala Ala Ile Thr Thr Ser Gly Asp Thr Thr Tyr Tyr Ala Glu Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala His Arg Gly Gly Gly Val Ile Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Xaa Xaa 115 <210> 16 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Made in lab - anti-CD33 VHH sequence <220> <221> MOD_RES <222> (120)..(121) <223> Xaa is any amino acid or absent <400> 16 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Glu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Phe Ser Ile Ser 20 25 30 Ile Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val 35 40 45 Ala Ser Thr Thr Ser Ser Gly Thr Thr Asn Tyr Val Glu Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys His 85 90 95 Ala Tyr Ile Ala Thr Thr Thr Asp Arg Gly Tyr Arg Gly Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Xaa Xaa 115 120 <210> 17 <211> 124 <212> PRT <213> Artificial Sequence <220> <223> Made in lab - anti-CD33 VHH sequence <220> <221> MOD_RES <222> (123)..(124) <223> Xaa is any amino acid or absent <400> 17 Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Ser Ser Ser Tyr Asn 20 25 30 Val Val Gly Trp Tyr Arg Gln Leu Ser Gly Asn Glu Arg Gly Gly Arg 35 40 45 Thr Met Val Ala Gln Ile Asn Ala Tyr Gly Asp Thr Asn Tyr Ala Asn 50 55 60 Ala Val Val Gly Arg Phe Thr Ile Ser Arg Asp Asp Ala Lys Asn Thr 65 70 75 80 Val Tyr Leu His Met Ser Asn Leu Lys Pro Glu Asp Thr Gly Val Tyr 85 90 95 Tyr Cys Asn Gly Gln Arg Met Leu Glu Asn Tyr Thr Tyr Arg Asp Gln 100 105 110 Ser Trp Gly Gln Gly Thr Gln Val Thr Val Xaa Xaa 115 120 <210> 18 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> Made in lab - anti-CD33 VHH sequence <220> <221> MOD_RES <222> (115)..(116) <223> Xaa is any amino acid or absent <400> 18 Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Thr Leu Ser Cys Ala Ala Ser Arg Ser Ser Gly Ile Asp Val 20 25 30 Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val Ala 35 40 45 Glu Ile Ser Gly Val Gly Asp Thr Asn Tyr Ala Ala Ser Leu Ala Asp 50 55 60 Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln 65 70 75 80 Met Lys Asn Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn Ala 85 90 95 His Ser Phe Leu Asp Leu Val Gly Ala Trp Gly Gln Gly Thr Gln Val 100 105 110 Thr Val Xaa Xaa 115 <210> 19 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Made in lab - anti-CD33 VHH sequence <220> <221> MOD_RES <222> (119)..(120) <223> Xaa is any amino acid or absent <400> 19 Glu Val Gln Leu Val Gln Ser Gly Gly Gly Ser Val Gln Val Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Leu Asn Ile Asp 20 25 30 His Ile Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val 35 40 45 Gly Val Ile Ser Ser Gly Ala Gly Pro Asn Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Asn Cys Asn 85 90 95 Ala Trp Ile Asp Tyr Gly Ser Gly Leu Pro Gln Asn Tyr Trp Gly Gln 100 105 110 Gly Thr Gln Val Thr Val Xaa Xaa 115 120 <210> 20 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> Made in lab - anti-CD33 VHH sequence <220> <221> MOD_RES <222> (115)..(116) <223> Xaa is any amino acid or absent <400> 20 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Glu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Arg Ser Ser Gly Ile Asp Val 20 25 30 Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val Ala 35 40 45 Glu Ile Ser Gly Val Gly Asp Thr Asn Tyr Ala Ala Ser Leu Ala Asp 50 55 60 Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln 65 70 75 80 Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Asn Ala 85 90 95 His Ser Phe Leu Asp Leu Val Gly Ala Trp Gly Gin Gly Thr Leu Val 100 105 110 Thr Val Xaa Xaa 115 <210> 21 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Made in lab - anti-CD33 VHH sequence <220> <221> MOD_RES <222> (119)..(120) <223> Xaa is any amino acid or absent <400> 21 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Glu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Leu Asn Ile Asp 20 25 30 His Ile Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val 35 40 45 Gly Val Ile Ser Ser Gly Ala Gly Pro Asn Tyr Ala Glu Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Asn 85 90 95 Ala Trp Ile Asp Tyr Gly Ser Gly Leu Pro Gln Asn Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Xaa Xaa 115 120 <210> 22 <211> 292 <212> PRT <213> Artificial Sequence <220> <223> Made in Lab - anti-CD33 VHH DARIC binding components <400> 22 Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Gln Val Thr Leu Arg Glu Ser Gly Gly Gly Leu Val 20 25 30 Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Lys Gly Ser Gly Arg Ala 35 40 45 Ile Asn Thr Tyr Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu 50 55 60 Arg Glu Phe Val Ala Ala Ile Ser Trp Asn Gly Gly His Thr Arg Tyr 65 70 75 80 Ala Asp Ser Val Gln Gly Arg Phe Ala Ile Ser Arg Asp Asn Ala Asp 85 90 95 Asn Thr Met Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala 100 105 110 Val Tyr His Cys Ala Ala Tyr Ser Asp Tyr His Arg Ile Ala Thr Met 115 120 125 Glu Ala Asp Ala Asp Ser Trp Gly Gly Thr Gly Thr Gln Val Thr Val Ser 130 135 140 Ser Gly Gly Gly Gly Ser Gly Val Gln Val Glu Thr Ile Ser Pro Gly 145 150 155 160 Asp Gly Arg Thr Phe Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr 165 170 175 Thr Gly Met Leu Glu Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg 180 185 190 Asn Lys Pro Phe Lys Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly 195 200 205 Trp Glu Glu Gly Val Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu 210 215 220 Thr Ile Ser Pro Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile 225 230 235 240 Ile Pro Pro His Ala Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu 245 250 255 Glu Gly Gly Arg Met Ala Leu Ile Val Leu Gly Gly Val Ala Gly Leu 260 265 270 Leu Leu Phe Ile Gly Leu Gly Ile Phe Phe Cys Val Arg Cys Arg His 275 280 285 Arg Arg Arg Gln 290 <210> 23 <211> 287 <212> PRT <213> Artificial Sequence <220> <223> Made in Lab - anti-CD33 VHH DARIC binding components <400> 23 Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Glu Val Gln Leu Val Glu Ser Gly Gly Gly Glu Val 20 25 30 Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr 35 40 45 Phe Ser Gly Tyr Ile Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu 50 55 60 Arg Glu Leu Val Ala Arg Ile Ser Gly Asn Asn Leu Ser Thr Glu Tyr 65 70 75 80 Ala Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys 85 90 95 Asn Thr Leu Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala 100 105 110 Val Tyr Tyr Cys Ala Ala Glu Tyr Asp Tyr Ser Ser Gly Asp Phe Val 115 120 125 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Lys Pro Gly Gly Gly Gly 130 135 140 Ser Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr Phe 145 150 155 160 Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr Thr Gly Met Leu Glu 165 170 175 Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe Lys 180 185 190 Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu Glu Gly Val 195 200 205 Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile Ser Pro Asp 210 215 220 Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile Ile Pro His Ala 225 230 235 240 Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu Glu Gly Gly Arg Met 245 250 255 Ala Leu Ile Val Leu Gly Gly Val Ala Gly Leu Leu Leu Phe Ile Gly 260 265 270 Leu Gly Ile Phe Phe Cys Val Arg Cys Arg His Arg Arg Arg Gln 275 280 285 <210> 24 <211> 285 <212> PRT <213> Artificial Sequence <220> <223> Made in Lab - anti-CD33 VHH DARIC binding components <400> 24 Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Glu Val Gln Leu Val Glu Ser Gly Gly Gly Glu Val 20 25 30 Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Gly 35 40 45 Phe Ser Ala Ser Leu Met Ser Trp His Arg Gln Ala Pro Gly Lys Gln 50 55 60 Arg Asp Leu Val Ala Ser Ile Thr Arg Asp Gly Arg Ala Asn Tyr Val 65 70 75 80 Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn 85 90 95 Thr Leu Tyr Leu Gln Met Ser Leu Arg Ala Glu Asp Thr Ala Val 100 105 110 Tyr Tyr Cys His Ala Tyr Ser Phe Asp Tyr Pro Ile Arg Ser Tyr Trp 115 120 125 Gly Gln Gly Thr Leu Val Thr Val Lys Pro Gly Gly Gly Gly Ser Gly 130 135 140 Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr Phe Pro Lys 145 150 155 160 Arg Gly Gln Thr Cys Val Val His Tyr Thr Gly Met Leu Glu Asp Gly 165 170 175 Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe Lys Phe Met 180 185 190 Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu Glu Gly Val Ala Gln 195 200 205 Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile Ser Pro Asp Tyr Ala 210 215 220 Tyr Gly Ala Thr Gly His Pro Gly Ile Ile Pro His Ala Thr Leu 225 230 235 240 Val Phe Asp Val Glu Leu Leu Lys Leu Glu Gly Gly Arg Met Ala Leu 245 250 255 Ile Val Leu Gly Gly Val Ala Gly Leu Leu Leu Phe Ile Gly Leu Gly 260 265 270 Ile Phe Phe Cys Val Arg Cys Arg His Arg Arg Arg Gln 275 280 285 <210> 25 <211> 285 <212> PRT <213> Artificial Sequence <220> <223> Made in Lab - anti-CD33 VHH DARIC binding components <400> 25 Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Glu Val Gln Leu Val Glu Ser Gly Gly Gly Glu Val 20 25 30 Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr 35 40 45 Phe Ser Ser Tyr Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly 50 55 60 Arg Glu Trp Val Ala Ala Ile Thr Thr Ser Gly Asp Thr Thr Tyr Tyr 65 70 75 80 Ala Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys 85 90 95 Asn Thr Val Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala 100 105 110 Val Tyr Tyr Cys Ala Ala His Arg Gly Gly Gly Val Ile Asp Tyr Trp 115 120 125 Gly Gln Gly Thr Leu Val Thr Val Lys Pro Gly Gly Gly Gly Ser Gly 130 135 140 Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr Phe Pro Lys 145 150 155 160 Arg Gly Gln Thr Cys Val Val His Tyr Thr Gly Met Leu Glu Asp Gly 165 170 175 Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe Lys Phe Met 180 185 190 Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu Glu Gly Val Ala Gln 195 200 205 Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile Ser Pro Asp Tyr Ala 210 215 220 Tyr Gly Ala Thr Gly His Pro Gly Ile Ile Pro His Ala Thr Leu 225 230 235 240 Val Phe Asp Val Glu Leu Leu Lys Leu Glu Gly Gly Arg Met Ala Leu 245 250 255 Ile Val Leu Gly Gly Val Ala Gly Leu Leu Leu Phe Ile Gly Leu Gly 260 265 270 Ile Phe Phe Cys Val Arg Cys Arg His Arg Arg Arg Gln 275 280 285 <210> 26 <211> 288 <212> PRT <213> Artificial Sequence <220> <223> Made in Lab - anti-CD33 VHH DARIC binding components <400> 26 Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Glu Val Gln Leu Val Glu Ser Gly Gly Gly Glu Val 20 25 30 Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile 35 40 45 Phe Ser Ile Ser Ile Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu 50 55 60 Arg Glu Leu Val Ala Ser Thr Thr Ser Ser Gly Thr Thr Asn Tyr Val 65 70 75 80 Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn 85 90 95 Thr Leu Tyr Leu Gln Met Ser Leu Arg Ala Glu Asp Thr Ala Val 100 105 110 Tyr Tyr Cys His Ala Tyr Ile Ala Thr Thr Thr Asp Arg Gly Tyr Arg 115 120 125 Gly Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Lys Pro Gly Gly Gly 130 135 140 Gly Ser Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr 145 150 155 160 Phe Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr Thr Gly Met Leu 165 170 175 Glu Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe 180 185 190 Lys Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu Glu Gly 195 200 205 Val Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile Ser Pro 210 215 220 Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile Ile Pro His 225 230 235 240 Ala Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu Glu Gly Gly Arg 245 250 255 Met Ala Leu Ile Val Leu Gly Gly Val Ala Gly Leu Leu Leu Phe Ile 260 265 270 Gly Leu Gly Ile Phe Phe Cys Val Arg Cys Arg His Arg Arg Arg Gln 275 280 285 <210> 27 <211> 291 <212> PRT <213> Artificial Sequence <220> <223> Made in Lab - anti-CD33 VHH DARIC binding components <400> 27 Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val 20 25 30 Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile 35 40 45 Ser Ser Tyr Asn Val Val Gly Trp Tyr Arg Gln Leu Ser Gly Asn Glu 50 55 60 Arg Gly Gly Arg Thr Met Val Ala Gln Ile Asn Ala Tyr Gly Asp Thr 65 70 75 80 Asn Tyr Ala Asn Ala Val Val Gly Arg Phe Thr Ile Ser Arg Asp Asp 85 90 95 Ala Lys Asn Thr Val Tyr Leu His Met Ser Asn Leu Lys Pro Glu Asp 100 105 110 Thr Gly Val Tyr Tyr Cys Asn Gly Gln Arg Met Leu Glu Asn Tyr Thr 115 120 125 Tyr Arg Asp Gln Ser Trp Gly Gly Gly Thr Gln Val Thr Val Lys Pro 130 135 140 Gly Gly Gly Gly Ser Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp 145 150 155 160 Gly Arg Thr Phe Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr Thr 165 170 175 Gly Met Leu Glu Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn 180 185 190 Lys Pro Phe Lys Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly Trp 195 200 205 Glu Glu Gly Val Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu Thr 210 215 220 Ile Ser Pro Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile Ile 225 230 235 240 Pro Pro His Ala Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu Glu 245 250 255 Gly Gly Arg Met Ala Leu Ile Val Leu Gly Gly Val Ala Gly Leu Leu 260 265 270 Leu Phe Ile Gly Leu Gly Ile Phe Phe Cys Val Arg Cys Arg His Arg 275 280 285 Arg Arg Gln 290 <210> 28 <211> 283 <212> PRT <213> Artificial Sequence <220> <223> Made in Lab - anti-CD33 VHH DARIC binding components <400> 28 Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val 20 25 30 Gln Ala Gly Gly Ser Leu Thr Leu Ser Cys Ala Ala Ser Arg Ser Ser 35 40 45 Gly Ile Asp Val Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg 50 55 60 Glu Leu Val Ala Glu Ile Ser Gly Val Gly Asp Thr Asn Tyr Ala Ala 65 70 75 80 Ser Leu Ala Asp Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Asn Thr 85 90 95 Val Tyr Leu Gln Met Lys Asn Leu Lys Pro Glu Asp Thr Ala Val Tyr 100 105 110 Tyr Cys Asn Ala His Ser Phe Leu Asp Leu Val Gly Ala Trp Gly Gln 115 120 125 Gly Thr Gln Val Thr Val Lys Pro Gly Gly Gly Gly Ser Gly Val Gln 130 135 140 Val Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr Phe Pro Lys Arg Gly 145 150 155 160 Gln Thr Cys Val Val His Tyr Thr Gly Met Leu Glu Asp Gly Lys Lys 165 170 175 Phe Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe Lys Phe Met Leu Gly 180 185 190 Lys Gln Glu Val Ile Arg Gly Trp Glu Glu Gly Val Ala Gln Met Ser 195 200 205 Val Gly Gln Arg Ala Lys Leu Thr Ile Ser Pro Asp Tyr Ala Tyr Gly 210 215 220 Ala Thr Gly His Pro Gly Ile Ile Pro His Ala Thr Leu Val Phe 225 230 235 240 Asp Val Glu Leu Leu Lys Leu Glu Gly Gly Arg Met Ala Leu Ile Val 245 250 255 Leu Gly Gly Val Ala Gly Leu Leu Leu Phe Ile Gly Leu Gly Ile Phe 260 265 270 Phe Cys Val Arg Cys Arg His Arg Arg Arg Gln 275 280 <210> 29 <211> 287 <212> PRT <213> Artificial Sequence <220> <223> Made in Lab - anti-CD33 VHH DARIC binding components <400> 29 Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Glu Val Gln Leu Val Gln Ser Gly Gly Gly Ser Val 20 25 30 Gln Val Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr 35 40 45 Leu Asn Ile Asp His Ile Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu 50 55 60 Arg Glu Leu Val Gly Val Ile Ser Ser Gly Ala Gly Pro Asn Tyr Ala 65 70 75 80 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn 85 90 95 Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val 100 105 110 Tyr Asn Cys Asn Ala Trp Ile Asp Tyr Gly Ser Gly Leu Pro Gln Asn 115 120 125 Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Lys Pro Gly Gly Gly Gly 130 135 140 Ser Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr Phe 145 150 155 160 Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr Thr Gly Met Leu Glu 165 170 175 Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe Lys 180 185 190 Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu Glu Gly Val 195 200 205 Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile Ser Pro Asp 210 215 220 Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile Ile Pro His Ala 225 230 235 240 Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu Glu Gly Gly Arg Met 245 250 255 Ala Leu Ile Val Leu Gly Gly Val Ala Gly Leu Leu Leu Phe Ile Gly 260 265 270 Leu Gly Ile Phe Phe Cys Val Arg Cys Arg His Arg Arg Arg Gln 275 280 285 <210> 30 <211> 283 <212> PRT <213> Artificial Sequence <220> <223> Made in Lab - anti-CD33 VHH DARIC binding components <400> 30 Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Glu Val Gln Leu Val Glu Ser Gly Gly Gly Glu Val 20 25 30 Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Arg Ser Ser 35 40 45 Gly Ile Asp Val Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg 50 55 60 Glu Leu Val Ala Glu Ile Ser Gly Val Gly Asp Thr Asn Tyr Ala Ala 65 70 75 80 Ser Leu Ala Asp Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Asn Thr 85 90 95 Val Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 100 105 110 Tyr Cys Asn Ala His Ser Phe Leu Asp Leu Val Gly Ala Trp Gly Gln 115 120 125 Gly Thr Leu Val Thr Val Lys Pro Gly Gly Gly Gly Ser Gly Val Gln 130 135 140 Val Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr Phe Pro Lys Arg Gly 145 150 155 160 Gln Thr Cys Val Val His Tyr Thr Gly Met Leu Glu Asp Gly Lys Lys 165 170 175 Phe Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe Lys Phe Met Leu Gly 180 185 190 Lys Gln Glu Val Ile Arg Gly Trp Glu Glu Gly Val Ala Gln Met Ser 195 200 205 Val Gly Gln Arg Ala Lys Leu Thr Ile Ser Pro Asp Tyr Ala Tyr Gly 210 215 220 Ala Thr Gly His Pro Gly Ile Ile Pro His Ala Thr Leu Val Phe 225 230 235 240 Asp Val Glu Leu Leu Lys Leu Glu Gly Gly Arg Met Ala Leu Ile Val 245 250 255 Leu Gly Gly Val Ala Gly Leu Leu Leu Phe Ile Gly Leu Gly Ile Phe 260 265 270 Phe Cys Val Arg Cys Arg His Arg Arg Arg Gln 275 280 <210> 31 <211> 287 <212> PRT <213> Artificial Sequence <220> <223> Made in Lab - anti-CD33 VHH DARIC binding components <400> 31 Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Glu Val Gln Leu Val Glu Ser Gly Gly Gly Glu Val 20 25 30 Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr 35 40 45 Leu Asn Ile Asp His Ile Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu 50 55 60 Arg Glu Leu Val Gly Val Ile Ser Ser Gly Ala Gly Pro Asn Tyr Ala 65 70 75 80 Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn 85 90 95 Thr Val Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Asn Ala Trp Ile Asp Tyr Gly Ser Gly Leu Pro Gln Asn 115 120 125 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Lys Pro Gly Gly Gly Gly 130 135 140 Ser Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr Phe 145 150 155 160 Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr Thr Gly Met Leu Glu 165 170 175 Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe Lys 180 185 190 Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu Glu Gly Val 195 200 205 Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile Ser Pro Asp 210 215 220 Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile Ile Pro His Ala 225 230 235 240 Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu Glu Gly Gly Arg Met 245 250 255 Ala Leu Ile Val Leu Gly Gly Val Ala Gly Leu Leu Leu Phe Ile Gly 260 265 270 Leu Gly Ile Phe Phe Cys Val Arg Cys Arg His Arg Arg Arg Gln 275 280 285 <210> 32 <211> 617 <212> PRT <213> Artificial Sequence <220> <223> Made in Lab - anti-CD33 VHH DARIC fusion protein <400> 32 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Gly Ser Ile Leu Trp His Glu Met Trp His Glu 20 25 30 Gly Leu Glu Glu Ala Ser Arg Leu Tyr Phe Gly Glu Arg Asn Val Lys 35 40 45 Gly Met Phe Glu Val Leu Glu Pro Leu His Ala Met Met Glu Arg Gly 50 55 60 Pro Gln Thr Leu Lys Glu Thr Ser Phe Asn Gln Ala Tyr Gly Arg Asp 65 70 75 80 Leu Met Glu Ala Gln Glu Trp Cys Arg Lys Tyr Met Lys Ser Gly Asn 85 90 95 Val Lys Asp Leu Leu Gln Ala Trp Asp Leu Tyr Tyr His Val Phe Arg 100 105 110 Arg Ile Ser Lys Ala Ser Ala Gly Thr Gly Ser Asp Ile Tyr Ile Trp 115 120 125 Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile 130 135 140 Thr Met His Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln 145 150 155 160 Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser 165 170 175 Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys 180 185 190 Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln 195 200 205 Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu 210 215 220 Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg 225 230 235 240 Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met 245 250 255 Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly 260 265 270 Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp 275 280 285 Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Ser Gly Ser 290 295 300 Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu 305 310 315 320 Asn Pro Gly Pro Ser Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu 325 330 335 Leu Leu Trp Val Pro Gly Ser Thr Gly Gin Val Thr Leu Arg Glu Ser 340 345 350 Gly Gly Gly Leu Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Lys 355 360 365 Gly Ser Gly Arg Ala Ile Asn Thr Tyr Ala Met Gly Trp Phe Arg Gln 370 375 380 Ala Pro Gly Lys Glu Arg Glu Phe Val Ala Ala Ile Ser Trp Asn Gly 385 390 395 400 Gly His Thr Arg Tyr Ala Asp Ser Val Gin Gly Arg Phe Ala Ile Ser 405 410 415 Arg Asp Asn Ala Asp Asn Thr Met Tyr Leu Gln Met Asn Ser Leu Lys 420 425 430 Pro Glu Asp Thr Ala Val Tyr His Cys Ala Ala Tyr Ser Asp Tyr His 435 440 445 Arg Ile Ala Thr Met Glu Ala Asp Ala Asp Ser Trp Gly Gln Gly Thr 450 455 460 Gln Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Val Gln Val Glu 465 470 475 480 Thr Ile Ser Pro Gly Asp Gly Arg Thr Phe Pro Lys Arg Gly Gln Thr 485 490 495 Cys Val Val His Tyr Thr Gly Met Leu Glu Asp Gly Lys Lys Phe Asp 500 505 510 Ser Ser Arg Asp Arg Asn Lys Pro Phe Lys Phe Met Leu Gly Lys Gln 515 520 525 Glu Val Ile Arg Gly Trp Glu Glu Gly Val Ala Gln Met Ser Val Gly 530 535 540 Gln Arg Ala Lys Leu Thr Ile Ser Pro Asp Tyr Ala Tyr Gly Ala Thr 545 550 555 560 Gly His Pro Gly Ile Ile Pro His Ala Thr Leu Val Phe Asp Val 565 570 575 Glu Leu Leu Lys Leu Glu Gly Gly Arg Met Ala Leu Ile Val Leu Gly 580 585 590 Gly Val Ala Gly Leu Leu Leu Phe Ile Gly Leu Gly Ile Phe Phe Cys 595 600 605 Val Arg Cys Arg His Arg Arg Arg Gln 610 615 <210> 33 <211> 612 <212> PRT <213> Artificial Sequence <220> <223> Made in Lab - anti-CD33 VHH DARIC fusion protein <400> 33 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Gly Ser Ile Leu Trp His Glu Met Trp His Glu 20 25 30 Gly Leu Glu Glu Ala Ser Arg Leu Tyr Phe Gly Glu Arg Asn Val Lys 35 40 45 Gly Met Phe Glu Val Leu Glu Pro Leu His Ala Met Met Glu Arg Gly 50 55 60 Pro Gln Thr Leu Lys Glu Thr Ser Phe Asn Gln Ala Tyr Gly Arg Asp 65 70 75 80 Leu Met Glu Ala Gln Glu Trp Cys Arg Lys Tyr Met Lys Ser Gly Asn 85 90 95 Val Lys Asp Leu Leu Gln Ala Trp Asp Leu Tyr Tyr His Val Phe Arg 100 105 110 Arg Ile Ser Lys Ala Ser Ala Gly Thr Gly Ser Asp Ile Tyr Ile Trp 115 120 125 Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile 130 135 140 Thr Met His Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln 145 150 155 160 Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser 165 170 175 Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys 180 185 190 Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln 195 200 205 Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu 210 215 220 Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg 225 230 235 240 Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met 245 250 255 Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly 260 265 270 Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp 275 280 285 Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Ser Gly Ser 290 295 300 Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu 305 310 315 320 Asn Pro Gly Pro Ser Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu 325 330 335 Leu Leu Trp Val Pro Gly Ser Thr Gly Glu Val Gln Leu Val Glu Ser 340 345 350 Gly Gly Gly Glu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala 355 360 365 Ala Ser Gly Arg Thr Phe Ser Gly Tyr Ile Met Gly Trp Phe Arg Gln 370 375 380 Ala Pro Gly Lys Glu Arg Glu Leu Val Ala Arg Ile Ser Gly Asn Asn 385 390 395 400 Leu Ser Thr Glu Tyr Ala Glu Ser Val Lys Gly Arg Phe Thr Ile Ser 405 410 415 Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Ser Ser Leu Arg 420 425 430 Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala Glu Tyr Asp Tyr Ser 435 440 445 Ser Gly Asp Phe Val Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Lys 450 455 460 Pro Gly Gly Gly Gly Ser Gly Val Gln Val Glu Thr Ile Ser Pro Gly 465 470 475 480 Asp Gly Arg Thr Phe Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr 485 490 495 Thr Gly Met Leu Glu Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg 500 505 510 Asn Lys Pro Phe Lys Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly 515 520 525 Trp Glu Glu Gly Val Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu 530 535 540 Thr Ile Ser Pro Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile 545 550 555 560 Ile Pro Pro His Ala Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu 565 570 575 Glu Gly Gly Arg Met Ala Leu Ile Val Leu Gly Gly Val Ala Gly Leu 580 585 590 Leu Leu Phe Ile Gly Leu Gly Ile Phe Phe Cys Val Arg Cys Arg His 595 600 605 Arg Arg Arg Gln 610 <210> 34 <211> 610 <212> PRT <213> Artificial Sequence <220> <223> Made in Lab - anti-CD33 VHH DARIC fusion protein <400> 34 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Gly Ser Ile Leu Trp His Glu Met Trp His Glu 20 25 30 Gly Leu Glu Glu Ala Ser Arg Leu Tyr Phe Gly Glu Arg Asn Val Lys 35 40 45 Gly Met Phe Glu Val Leu Glu Pro Leu His Ala Met Met Glu Arg Gly 50 55 60 Pro Gln Thr Leu Lys Glu Thr Ser Phe Asn Gln Ala Tyr Gly Arg Asp 65 70 75 80 Leu Met Glu Ala Gln Glu Trp Cys Arg Lys Tyr Met Lys Ser Gly Asn 85 90 95 Val Lys Asp Leu Leu Gln Ala Trp Asp Leu Tyr Tyr His Val Phe Arg 100 105 110 Arg Ile Ser Lys Ala Ser Ala Gly Thr Gly Ser Asp Ile Tyr Ile Trp 115 120 125 Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile 130 135 140 Thr Met His Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln 145 150 155 160 Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser 165 170 175 Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys 180 185 190 Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln 195 200 205 Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu 210 215 220 Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg 225 230 235 240 Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met 245 250 255 Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly 260 265 270 Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp 275 280 285 Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Ser Gly Ser 290 295 300 Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu 305 310 315 320 Asn Pro Gly Pro Ser Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu 325 330 335 Leu Leu Trp Val Pro Gly Ser Thr Gly Glu Val Gln Leu Val Glu Ser 340 345 350 Gly Gly Gly Glu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala 355 360 365 Ala Ser Gly Ser Gly Phe Ser Ala Ser Leu Met Ser Trp His Arg Gln 370 375 380 Ala Pro Gly Lys Gln Arg Asp Leu Val Ala Ser Ile Thr Arg Asp Gly 385 390 395 400 Arg Ala Asn Tyr Val Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg 405 410 415 Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Ser Ser Leu Arg Ala 420 425 430 Glu Asp Thr Ala Val Tyr Tyr Cys His Ala Tyr Ser Phe Asp Tyr Pro 435 440 445 Ile Arg Ser Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Lys Pro Gly 450 455 460 Gly Gly Gly Ser Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly 465 470 475 480 Arg Thr Phe Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr Thr Gly 485 490 495 Met Leu Glu Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn Lys 500 505 510 Pro Phe Lys Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu 515 520 525 Glu Gly Val Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile 530 535 540 Ser Pro Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile Ile Pro 545 550 555 560 Pro His Ala Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu Glu Gly 565 570 575 Gly Arg Met Ala Leu Ile Val Leu Gly Gly Val Ala Gly Leu Leu Leu 580 585 590 Phe Ile Gly Leu Gly Ile Phe Phe Cys Val Arg Cys Arg His Arg Arg 595 600 605 Arg Gln 610 <210> 35 <211> 610 <212> PRT <213> Artificial Sequence <220> <223> Made in Lab - anti-CD33 VHH DARIC fusion protein <400> 35 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Gly Ser Ile Leu Trp His Glu Met Trp His Glu 20 25 30 Gly Leu Glu Glu Ala Ser Arg Leu Tyr Phe Gly Glu Arg Asn Val Lys 35 40 45 Gly Met Phe Glu Val Leu Glu Pro Leu His Ala Met Met Glu Arg Gly 50 55 60 Pro Gln Thr Leu Lys Glu Thr Ser Phe Asn Gln Ala Tyr Gly Arg Asp 65 70 75 80 Leu Met Glu Ala Gln Glu Trp Cys Arg Lys Tyr Met Lys Ser Gly Asn 85 90 95 Val Lys Asp Leu Leu Gln Ala Trp Asp Leu Tyr Tyr His Val Phe Arg 100 105 110 Arg Ile Ser Lys Ala Ser Ala Gly Thr Gly Ser Asp Ile Tyr Ile Trp 115 120 125 Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile 130 135 140 Thr Met His Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln 145 150 155 160 Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser 165 170 175 Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys 180 185 190 Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln 195 200 205 Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu 210 215 220 Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg 225 230 235 240 Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met 245 250 255 Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly 260 265 270 Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp 275 280 285 Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Ser Gly Ser 290 295 300 Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu 305 310 315 320 Asn Pro Gly Pro Ser Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu 325 330 335 Leu Leu Trp Val Pro Gly Ser Thr Gly Glu Val Gln Leu Val Glu Ser 340 345 350 Gly Gly Gly Glu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala 355 360 365 Ala Ser Gly Phe Thr Phe Ser Ser Tyr Ala Met Gly Trp Phe Arg Gln 370 375 380 Ala Pro Gly Lys Gly Arg Glu Trp Val Ala Ala Ile Thr Thr Ser Gly 385 390 395 400 Asp Thr Thr Tyr Tyr Ala Glu Ser Val Lys Gly Arg Phe Thr Ile Ser 405 410 415 Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Ser Ser Leu Arg 420 425 430 Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala His Arg Gly Gly Gly 435 440 445 Val Ile Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Lys Pro Gly 450 455 460 Gly Gly Gly Ser Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly 465 470 475 480 Arg Thr Phe Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr Thr Gly 485 490 495 Met Leu Glu Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn Lys 500 505 510 Pro Phe Lys Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu 515 520 525 Glu Gly Val Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile 530 535 540 Ser Pro Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile Ile Pro 545 550 555 560 Pro His Ala Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu Glu Gly 565 570 575 Gly Arg Met Ala Leu Ile Val Leu Gly Gly Val Ala Gly Leu Leu Leu 580 585 590 Phe Ile Gly Leu Gly Ile Phe Phe Cys Val Arg Cys Arg His Arg Arg 595 600 605 Arg Gln 610 <210> 36 <211> 613 <212> PRT <213> Artificial Sequence <220> <223> Made in Lab - anti-CD33 VHH DARIC fusion protein <400> 36 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Gly Ser Ile Leu Trp His Glu Met Trp His Glu 20 25 30 Gly Leu Glu Glu Ala Ser Arg Leu Tyr Phe Gly Glu Arg Asn Val Lys 35 40 45 Gly Met Phe Glu Val Leu Glu Pro Leu His Ala Met Met Glu Arg Gly 50 55 60 Pro Gln Thr Leu Lys Glu Thr Ser Phe Asn Gln Ala Tyr Gly Arg Asp 65 70 75 80 Leu Met Glu Ala Gln Glu Trp Cys Arg Lys Tyr Met Lys Ser Gly Asn 85 90 95 Val Lys Asp Leu Leu Gln Ala Trp Asp Leu Tyr Tyr His Val Phe Arg 100 105 110 Arg Ile Ser Lys Ala Ser Ala Gly Thr Gly Ser Asp Ile Tyr Ile Trp 115 120 125 Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile 130 135 140 Thr Met His Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln 145 150 155 160 Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser 165 170 175 Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys 180 185 190 Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln 195 200 205 Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu 210 215 220 Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg 225 230 235 240 Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met 245 250 255 Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly 260 265 270 Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp 275 280 285 Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Ser Gly Ser 290 295 300 Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu 305 310 315 320 Asn Pro Gly Pro Ser Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu 325 330 335 Leu Leu Trp Val Pro Gly Ser Thr Gly Glu Val Gln Leu Val Glu Ser 340 345 350 Gly Gly Gly Glu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala 355 360 365 Ala Ser Gly Ser Ile Phe Ser Ile Ser Ile Met Gly Trp Tyr Arg Gln 370 375 380 Ala Pro Gly Lys Glu Arg Glu Leu Val Ala Ser Thr Thr Ser Ser Gly 385 390 395 400 Thr Thr Asn Tyr Val Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg 405 410 415 Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Ser Ser Leu Arg Ala 420 425 430 Glu Asp Thr Ala Val Tyr Tyr Cys His Ala Tyr Ile Ala Thr Thr Thr 435 440 445 Asp Arg Gly Tyr Arg Gly Tyr Trp Gly Gln Gly Thr Leu Val Thr Val 450 455 460 Lys Pro Gly Gly Gly Gly Ser Gly Val Gln Val Glu Thr Ile Ser Pro 465 470 475 480 Gly Asp Gly Arg Thr Phe Pro Lys Arg Gly Gln Thr Cys Val Val His 485 490 495 Tyr Thr Gly Met Leu Glu Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp 500 505 510 Arg Asn Lys Pro Phe Lys Phe Met Leu Gly Lys Gln Glu Val Ile Arg 515 520 525 Gly Trp Glu Glu Gly Val Ala Gln Met Ser Val Gly Gln Arg Ala Lys 530 535 540 Leu Thr Ile Ser Pro Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly 545 550 555 560 Ile Ile Pro His Ala Thr Leu Val Phe Asp Val Glu Leu Leu Lys 565 570 575 Leu Glu Gly Gly Arg Met Ala Leu Ile Val Leu Gly Gly Val Ala Gly 580 585 590 Leu Leu Leu Phe Ile Gly Leu Gly Ile Phe Phe Cys Val Arg Cys Arg 595 600 605 His Arg Arg Arg Gln 610 <210> 37 <211> 616 <212> PRT <213> Artificial Sequence <220> <223> Made in Lab - anti-CD33 VHH DARIC fusion protein <400> 37 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Gly Ser Ile Leu Trp His Glu Met Trp His Glu 20 25 30 Gly Leu Glu Glu Ala Ser Arg Leu Tyr Phe Gly Glu Arg Asn Val Lys 35 40 45 Gly Met Phe Glu Val Leu Glu Pro Leu His Ala Met Met Glu Arg Gly 50 55 60 Pro Gln Thr Leu Lys Glu Thr Ser Phe Asn Gln Ala Tyr Gly Arg Asp 65 70 75 80 Leu Met Glu Ala Gln Glu Trp Cys Arg Lys Tyr Met Lys Ser Gly Asn 85 90 95 Val Lys Asp Leu Leu Gln Ala Trp Asp Leu Tyr Tyr His Val Phe Arg 100 105 110 Arg Ile Ser Lys Ala Ser Ala Gly Thr Gly Ser Asp Ile Tyr Ile Trp 115 120 125 Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile 130 135 140 Thr Met His Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln 145 150 155 160 Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser 165 170 175 Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys 180 185 190 Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln 195 200 205 Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu 210 215 220 Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg 225 230 235 240 Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met 245 250 255 Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly 260 265 270 Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp 275 280 285 Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Ser Gly Ser 290 295 300 Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu 305 310 315 320 Asn Pro Gly Pro Ser Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu 325 330 335 Leu Leu Trp Val Pro Gly Ser Thr Gly Gln Val Gln Leu Val Gln Ser 340 345 350 Gly Gly Gly Leu Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Ala 355 360 365 Ala Ser Gly Ser Ile Ser Ser Tyr Asn Val Val Gly Trp Tyr Arg Gln 370 375 380 Leu Ser Gly Asn Glu Arg Gly Gly Arg Thr Met Val Ala Gln Ile Asn 385 390 395 400 Ala Tyr Gly Asp Thr Asn Tyr Ala Asn Ala Val Val Gly Arg Phe Thr 405 410 415 Ile Ser Arg Asp Asp Ala Lys Asn Thr Val Tyr Leu His Met Ser Asn 420 425 430 Leu Lys Pro Glu Asp Thr Gly Val Tyr Tyr Cys Asn Gly Gln Arg Met 435 440 445 Leu Glu Asn Tyr Thr Tyr Arg Asp Gln Ser Trp Gly Gln Gly Thr Gln 450 455 460 Val Thr Val Lys Pro Gly Gly Gly Gly Ser Gly Val Gln Val Glu Thr 465 470 475 480 Ile Ser Pro Gly Asp Gly Arg Thr Phe Pro Lys Arg Gly Gln Thr Cys 485 490 495 Val Val His Tyr Thr Gly Met Leu Glu Asp Gly Lys Lys Phe Asp Ser 500 505 510 Ser Arg Asp Arg Asn Lys Pro Phe Lys Phe Met Leu Gly Lys Gln Glu 515 520 525 Val Ile Arg Gly Trp Glu Glu Gly Val Ala Gln Met Ser Val Gly Gln 530 535 540 Arg Ala Lys Leu Thr Ile Ser Pro Asp Tyr Ala Tyr Gly Ala Thr Gly 545 550 555 560 His Pro Gly Ile Ile Pro His Ala Thr Leu Val Phe Asp Val Glu 565 570 575 Leu Leu Lys Leu Glu Gly Gly Arg Met Ala Leu Ile Val Leu Gly Gly 580 585 590 Val Ala Gly Leu Leu Leu Phe Ile Gly Leu Gly Ile Phe Phe Cys Val 595 600 605 Arg Cys Arg His Arg Arg Arg Gln 610 615 <210> 38 <211> 608 <212> PRT <213> Artificial Sequence <220> <223> Made in Lab - anti-CD33 VHH DARIC fusion protein <400> 38 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Gly Ser Ile Leu Trp His Glu Met Trp His Glu 20 25 30 Gly Leu Glu Glu Ala Ser Arg Leu Tyr Phe Gly Glu Arg Asn Val Lys 35 40 45 Gly Met Phe Glu Val Leu Glu Pro Leu His Ala Met Met Glu Arg Gly 50 55 60 Pro Gln Thr Leu Lys Glu Thr Ser Phe Asn Gln Ala Tyr Gly Arg Asp 65 70 75 80 Leu Met Glu Ala Gln Glu Trp Cys Arg Lys Tyr Met Lys Ser Gly Asn 85 90 95 Val Lys Asp Leu Leu Gln Ala Trp Asp Leu Tyr Tyr His Val Phe Arg 100 105 110 Arg Ile Ser Lys Ala Ser Ala Gly Thr Gly Ser Asp Ile Tyr Ile Trp 115 120 125 Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile 130 135 140 Thr Met His Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln 145 150 155 160 Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser 165 170 175 Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys 180 185 190 Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln 195 200 205 Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu 210 215 220 Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg 225 230 235 240 Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met 245 250 255 Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly 260 265 270 Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp 275 280 285 Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Ser Gly Ser 290 295 300 Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu 305 310 315 320 Asn Pro Gly Pro Ser Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu 325 330 335 Leu Leu Trp Val Pro Gly Ser Thr Gly Gln Val Gln Leu Val Gln Ser 340 345 350 Gly Gly Gly Leu Val Gln Ala Gly Gly Ser Leu Thr Leu Ser Cys Ala 355 360 365 Ala Ser Arg Ser Ser Gly Ile Asp Val Met Gly Trp Tyr Arg Gln Ala 370 375 380 Pro Gly Lys Glu Arg Glu Leu Val Ala Glu Ile Ser Gly Val Gly Asp 385 390 395 400 Thr Asn Tyr Ala Ala Ser Leu Ala Asp Arg Phe Thr Val Ser Arg Asp 405 410 415 Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Lys Asn Leu Lys Pro Glu 420 425 430 Asp Thr Ala Val Tyr Tyr Cys Asn Ala His Ser Phe Leu Asp Leu Val 435 440 445 Gly Ala Trp Gly Gln Gly Thr Gln Val Thr Val Lys Pro Gly Gly Gly 450 455 460 Gly Ser Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr 465 470 475 480 Phe Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr Thr Gly Met Leu 485 490 495 Glu Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe 500 505 510 Lys Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu Glu Gly 515 520 525 Val Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile Ser Pro 530 535 540 Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile Ile Pro His 545 550 555 560 Ala Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu Glu Gly Gly Arg 565 570 575 Met Ala Leu Ile Val Leu Gly Gly Val Ala Gly Leu Leu Leu Phe Ile 580 585 590 Gly Leu Gly Ile Phe Phe Cys Val Arg Cys Arg His Arg Arg Arg Gln 595 600 605 <210> 39 <211> 612 <212> PRT <213> Artificial Sequence <220> <223> Made in Lab - anti-CD33 VHH DARIC fusion protein <400> 39 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Gly Ser Ile Leu Trp His Glu Met Trp His Glu 20 25 30 Gly Leu Glu Glu Ala Ser Arg Leu Tyr Phe Gly Glu Arg Asn Val Lys 35 40 45 Gly Met Phe Glu Val Leu Glu Pro Leu His Ala Met Met Glu Arg Gly 50 55 60 Pro Gln Thr Leu Lys Glu Thr Ser Phe Asn Gln Ala Tyr Gly Arg Asp 65 70 75 80 Leu Met Glu Ala Gln Glu Trp Cys Arg Lys Tyr Met Lys Ser Gly Asn 85 90 95 Val Lys Asp Leu Leu Gln Ala Trp Asp Leu Tyr Tyr His Val Phe Arg 100 105 110 Arg Ile Ser Lys Ala Ser Ala Gly Thr Gly Ser Asp Ile Tyr Ile Trp 115 120 125 Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile 130 135 140 Thr Met His Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln 145 150 155 160 Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser 165 170 175 Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys 180 185 190 Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln 195 200 205 Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu 210 215 220 Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg 225 230 235 240 Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met 245 250 255 Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly 260 265 270 Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp 275 280 285 Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Ser Gly Ser 290 295 300 Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu 305 310 315 320 Asn Pro Gly Pro Ser Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu 325 330 335 Leu Leu Trp Val Pro Gly Ser Thr Gly Glu Val Gln Leu Val Gln Ser 340 345 350 Gly Gly Gly Ser Val Gln Val Gly Gly Ser Leu Arg Leu Ser Cys Ala 355 360 365 Ala Ser Gly Ser Thr Leu Asn Ile Asp His Ile Gly Trp Tyr Arg Gln 370 375 380 Ala Pro Gly Lys Glu Arg Glu Leu Val Gly Val Ile Ser Ser Gly Ala 385 390 395 400 Gly Pro Asn Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg 405 410 415 Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro 420 425 430 Glu Asp Thr Ala Val Tyr Asn Cys Asn Ala Trp Ile Asp Tyr Gly Ser 435 440 445 Gly Leu Pro Gln Asn Tyr Trp Gly Gly Gly Thr Gln Val Thr Val Lys 450 455 460 Pro Gly Gly Gly Gly Ser Gly Val Gln Val Glu Thr Ile Ser Pro Gly 465 470 475 480 Asp Gly Arg Thr Phe Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr 485 490 495 Thr Gly Met Leu Glu Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg 500 505 510 Asn Lys Pro Phe Lys Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly 515 520 525 Trp Glu Glu Gly Val Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu 530 535 540 Thr Ile Ser Pro Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile 545 550 555 560 Ile Pro Pro His Ala Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu 565 570 575 Glu Gly Gly Arg Met Ala Leu Ile Val Leu Gly Gly Val Ala Gly Leu 580 585 590 Leu Leu Phe Ile Gly Leu Gly Ile Phe Phe Cys Val Arg Cys Arg His 595 600 605 Arg Arg Arg Gln 610 <210> 40 <211> 608 <212> PRT <213> Artificial Sequence <220> <223> Made in Lab - anti-CD33 VHH DARIC fusion protein <400> 40 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Gly Ser Ile Leu Trp His Glu Met Trp His Glu 20 25 30 Gly Leu Glu Glu Ala Ser Arg Leu Tyr Phe Gly Glu Arg Asn Val Lys 35 40 45 Gly Met Phe Glu Val Leu Glu Pro Leu His Ala Met Met Glu Arg Gly 50 55 60 Pro Gln Thr Leu Lys Glu Thr Ser Phe Asn Gln Ala Tyr Gly Arg Asp 65 70 75 80 Leu Met Glu Ala Gln Glu Trp Cys Arg Lys Tyr Met Lys Ser Gly Asn 85 90 95 Val Lys Asp Leu Leu Gln Ala Trp Asp Leu Tyr Tyr His Val Phe Arg 100 105 110 Arg Ile Ser Lys Ala Ser Ala Gly Thr Gly Ser Asp Ile Tyr Ile Trp 115 120 125 Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile 130 135 140 Thr Met His Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln 145 150 155 160 Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser 165 170 175 Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys 180 185 190 Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln 195 200 205 Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu 210 215 220 Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg 225 230 235 240 Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met 245 250 255 Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly 260 265 270 Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp 275 280 285 Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Ser Gly Ser 290 295 300 Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu 305 310 315 320 Asn Pro Gly Pro Ser Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu 325 330 335 Leu Leu Trp Val Pro Gly Ser Thr Gly Glu Val Gln Leu Val Glu Ser 340 345 350 Gly Gly Gly Glu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala 355 360 365 Ala Ser Arg Ser Ser Gly Ile Asp Val Met Gly Trp Tyr Arg Gln Ala 370 375 380 Pro Gly Lys Glu Arg Glu Leu Val Ala Glu Ile Ser Gly Val Gly Asp 385 390 395 400 Thr Asn Tyr Ala Ala Ser Leu Ala Asp Arg Phe Thr Val Ser Arg Asp 405 410 415 Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu 420 425 430 Asp Thr Ala Val Tyr Tyr Cys Asn Ala His Ser Phe Leu Asp Leu Val 435 440 445 Gly Ala Trp Gly Gln Gly Thr Leu Val Thr Val Lys Pro Gly Gly Gly 450 455 460 Gly Ser Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr 465 470 475 480 Phe Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr Thr Gly Met Leu 485 490 495 Glu Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe 500 505 510 Lys Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu Glu Gly 515 520 525 Val Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile Ser Pro 530 535 540 Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile Ile Pro His 545 550 555 560 Ala Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu Glu Gly Gly Arg 565 570 575 Met Ala Leu Ile Val Leu Gly Gly Val Ala Gly Leu Leu Leu Phe Ile 580 585 590 Gly Leu Gly Ile Phe Phe Cys Val Arg Cys Arg His Arg Arg Arg Gln 595 600 605 <210> 41 <211> 612 <212> PRT <213> Artificial Sequence <220> <223> Made in Lab - anti-CD33 VHH DARIC fusion protein <400> 41 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Gly Ser Ile Leu Trp His Glu Met Trp His Glu 20 25 30 Gly Leu Glu Glu Ala Ser Arg Leu Tyr Phe Gly Glu Arg Asn Val Lys 35 40 45 Gly Met Phe Glu Val Leu Glu Pro Leu His Ala Met Met Glu Arg Gly 50 55 60 Pro Gln Thr Leu Lys Glu Thr Ser Phe Asn Gln Ala Tyr Gly Arg Asp 65 70 75 80 Leu Met Glu Ala Gln Glu Trp Cys Arg Lys Tyr Met Lys Ser Gly Asn 85 90 95 Val Lys Asp Leu Leu Gln Ala Trp Asp Leu Tyr Tyr His Val Phe Arg 100 105 110 Arg Ile Ser Lys Ala Ser Ala Gly Thr Gly Ser Asp Ile Tyr Ile Trp 115 120 125 Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile 130 135 140 Thr Met His Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln 145 150 155 160 Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser 165 170 175 Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys 180 185 190 Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln 195 200 205 Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu 210 215 220 Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg 225 230 235 240 Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met 245 250 255 Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly 260 265 270 Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp 275 280 285 Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Ser Gly Ser 290 295 300 Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu 305 310 315 320 Asn Pro Gly Pro Ser Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu 325 330 335 Leu Leu Trp Val Pro Gly Ser Thr Gly Glu Val Gln Leu Val Glu Ser 340 345 350 Gly Gly Gly Glu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala 355 360 365 Ala Ser Gly Ser Thr Leu Asn Ile Asp His Ile Gly Trp Tyr Arg Gln 370 375 380 Ala Pro Gly Lys Glu Arg Glu Leu Val Gly Val Ile Ser Ser Gly Ala 385 390 395 400 Gly Pro Asn Tyr Ala Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg 405 410 415 Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Ser Ser Leu Arg Ala 420 425 430 Glu Asp Thr Ala Val Tyr Tyr Cys Asn Ala Trp Ile Asp Tyr Gly Ser 435 440 445 Gly Leu Pro Gln Asn Tyr Trp Gly Gly Gly Thr Leu Val Thr Val Lys 450 455 460 Pro Gly Gly Gly Gly Ser Gly Val Gln Val Glu Thr Ile Ser Pro Gly 465 470 475 480 Asp Gly Arg Thr Phe Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr 485 490 495 Thr Gly Met Leu Glu Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg 500 505 510 Asn Lys Pro Phe Lys Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly 515 520 525 Trp Glu Glu Gly Val Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu 530 535 540 Thr Ile Ser Pro Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile 545 550 555 560 Ile Pro Pro His Ala Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu 565 570 575 Glu Gly Gly Arg Met Ala Leu Ile Val Leu Gly Gly Val Ala Gly Leu 580 585 590 Leu Leu Phe Ile Gly Leu Gly Ile Phe Phe Cys Val Arg Cys Arg His 595 600 605 Arg Arg Arg Gln 610 <210> 42 <211> 649 <212> PRT <213> Artificial Sequence <220> <223> Made in Lab - anti-CD33 VHH DARIC.OX40 fusion protein <400> 42 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Gly Ser Ile Leu Trp His Glu Met Trp His Glu 20 25 30 Gly Leu Glu Glu Ala Ser Arg Leu Tyr Phe Gly Glu Arg Asn Val Lys 35 40 45 Gly Met Phe Glu Val Leu Glu Pro Leu His Ala Met Met Glu Arg Gly 50 55 60 Pro Gln Thr Leu Lys Glu Thr Ser Phe Asn Gln Ala Tyr Gly Arg Asp 65 70 75 80 Leu Met Glu Ala Gln Glu Trp Cys Arg Lys Tyr Met Lys Ser Gly Asn 85 90 95 Val Lys Asp Leu Leu Gln Ala Trp Asp Leu Tyr Tyr His Val Phe Arg 100 105 110 Arg Ile Ser Lys Ala Ser Ala Gly Thr Gly Ser Asp Ile Tyr Ile Trp 115 120 125 Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile 130 135 140 Thr Met His Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln 145 150 155 160 Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser 165 170 175 Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys 180 185 190 Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln 195 200 205 Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu 210 215 220 Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg 225 230 235 240 Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met 245 250 255 Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly 260 265 270 Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp 275 280 285 Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Ser Gly Ser 290 295 300 Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu 305 310 315 320 Asn Pro Gly Pro Ser Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu 325 330 335 Leu Leu Trp Val Pro Gly Ser Thr Gly Gin Val Thr Leu Arg Glu Ser 340 345 350 Gly Gly Gly Leu Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Lys 355 360 365 Gly Ser Gly Arg Ala Ile Asn Thr Tyr Ala Met Gly Trp Phe Arg Gln 370 375 380 Ala Pro Gly Lys Glu Arg Glu Phe Val Ala Ala Ile Ser Trp Asn Gly 385 390 395 400 Gly His Thr Arg Tyr Ala Asp Ser Val Gin Gly Arg Phe Ala Ile Ser 405 410 415 Arg Asp Asn Ala Asp Asn Thr Met Tyr Leu Gln Met Asn Ser Leu Lys 420 425 430 Pro Glu Asp Thr Ala Val Tyr His Cys Ala Ala Tyr Ser Asp Tyr His 435 440 445 Arg Ile Ala Thr Met Glu Ala Asp Ala Asp Ser Trp Gly Gln Gly Thr 450 455 460 Gln Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Val Gln Val Glu 465 470 475 480 Thr Ile Ser Pro Gly Asp Gly Arg Thr Phe Pro Lys Arg Gly Gln Thr 485 490 495 Cys Val Val His Tyr Thr Gly Met Leu Glu Asp Gly Lys Lys Phe Asp 500 505 510 Ser Ser Arg Asp Arg Asn Lys Pro Phe Lys Phe Met Leu Gly Lys Gln 515 520 525 Glu Val Ile Arg Gly Trp Glu Glu Gly Val Ala Gln Met Ser Val Gly 530 535 540 Gln Arg Ala Lys Leu Thr Ile Ser Pro Asp Tyr Ala Tyr Gly Ala Thr 545 550 555 560 Gly His Pro Gly Ile Ile Pro His Ala Thr Leu Val Phe Asp Val 565 570 575 Glu Leu Leu Lys Leu Glu Gly Gly Arg Met Ala Leu Ile Val Leu Gly 580 585 590 Gly Val Ala Gly Leu Leu Leu Phe Ile Gly Leu Gly Ile Phe Phe Ala 595 600 605 Leu Tyr Leu Leu Arg Arg Asp Gln Arg Leu Pro Pro Asp Ala His Lys 610 615 620 Pro Pro Gly Gly Gly Ser Phe Arg Thr Pro Ile Gln Glu Glu Gln Ala 625 630 635 640 Asp Ala His Ser Thr Leu Ala Lys Ile 645 <210> 43 <211> 644 <212> PRT <213> Artificial Sequence <220> <223> Made in Lab - anti-CD33 VHH DARIC.OX40 fusion protein <400> 43 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Gly Ser Ile Leu Trp His Glu Met Trp His Glu 20 25 30 Gly Leu Glu Glu Ala Ser Arg Leu Tyr Phe Gly Glu Arg Asn Val Lys 35 40 45 Gly Met Phe Glu Val Leu Glu Pro Leu His Ala Met Met Glu Arg Gly 50 55 60 Pro Gln Thr Leu Lys Glu Thr Ser Phe Asn Gln Ala Tyr Gly Arg Asp 65 70 75 80 Leu Met Glu Ala Gln Glu Trp Cys Arg Lys Tyr Met Lys Ser Gly Asn 85 90 95 Val Lys Asp Leu Leu Gln Ala Trp Asp Leu Tyr Tyr His Val Phe Arg 100 105 110 Arg Ile Ser Lys Ala Ser Ala Gly Thr Gly Ser Asp Ile Tyr Ile Trp 115 120 125 Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile 130 135 140 Thr Met His Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln 145 150 155 160 Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser 165 170 175 Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys 180 185 190 Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln 195 200 205 Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu 210 215 220 Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg 225 230 235 240 Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met 245 250 255 Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly 260 265 270 Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp 275 280 285 Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Ser Gly Ser 290 295 300 Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu 305 310 315 320 Asn Pro Gly Pro Ser Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu 325 330 335 Leu Leu Trp Val Pro Gly Ser Thr Gly Glu Val Gln Leu Val Glu Ser 340 345 350 Gly Gly Gly Glu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala 355 360 365 Ala Ser Gly Arg Thr Phe Ser Gly Tyr Ile Met Gly Trp Phe Arg Gln 370 375 380 Ala Pro Gly Lys Glu Arg Glu Leu Val Ala Arg Ile Ser Gly Asn Asn 385 390 395 400 Leu Ser Thr Glu Tyr Ala Glu Ser Val Lys Gly Arg Phe Thr Ile Ser 405 410 415 Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Ser Ser Leu Arg 420 425 430 Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala Glu Tyr Asp Tyr Ser 435 440 445 Ser Gly Asp Phe Val Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Lys 450 455 460 Pro Gly Gly Gly Gly Ser Gly Val Gln Val Glu Thr Ile Ser Pro Gly 465 470 475 480 Asp Gly Arg Thr Phe Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr 485 490 495 Thr Gly Met Leu Glu Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg 500 505 510 Asn Lys Pro Phe Lys Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly 515 520 525 Trp Glu Glu Gly Val Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu 530 535 540 Thr Ile Ser Pro Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile 545 550 555 560 Ile Pro Pro His Ala Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu 565 570 575 Glu Gly Gly Arg Met Ala Leu Ile Val Leu Gly Gly Val Ala Gly Leu 580 585 590 Leu Leu Phe Ile Gly Leu Gly Ile Phe Phe Ala Leu Tyr Leu Leu Arg 595 600 605 Arg Asp Gln Arg Leu Pro Pro Asp Ala His Lys Pro Pro Gly Gly Gly 610 615 620 Ser Phe Arg Thr Pro Ile Gln Glu Glu Gln Ala Asp Ala His Ser Thr 625 630 635 640 Leu Ala Lys Ile <210> 44 <211> 642 <212> PRT <213> Artificial Sequence <220> <223> Made in Lab - anti-CD33 VHH DARIC.OX40 fusion protein <400> 44 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Gly Ser Ile Leu Trp His Glu Met Trp His Glu 20 25 30 Gly Leu Glu Glu Ala Ser Arg Leu Tyr Phe Gly Glu Arg Asn Val Lys 35 40 45 Gly Met Phe Glu Val Leu Glu Pro Leu His Ala Met Met Glu Arg Gly 50 55 60 Pro Gln Thr Leu Lys Glu Thr Ser Phe Asn Gln Ala Tyr Gly Arg Asp 65 70 75 80 Leu Met Glu Ala Gln Glu Trp Cys Arg Lys Tyr Met Lys Ser Gly Asn 85 90 95 Val Lys Asp Leu Leu Gln Ala Trp Asp Leu Tyr Tyr His Val Phe Arg 100 105 110 Arg Ile Ser Lys Ala Ser Ala Gly Thr Gly Ser Asp Ile Tyr Ile Trp 115 120 125 Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile 130 135 140 Thr Met His Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln 145 150 155 160 Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser 165 170 175 Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys 180 185 190 Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln 195 200 205 Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu 210 215 220 Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg 225 230 235 240 Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met 245 250 255 Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly 260 265 270 Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp 275 280 285 Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Ser Gly Ser 290 295 300 Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu 305 310 315 320 Asn Pro Gly Pro Ser Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu 325 330 335 Leu Leu Trp Val Pro Gly Ser Thr Gly Glu Val Gln Leu Val Glu Ser 340 345 350 Gly Gly Gly Glu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala 355 360 365 Ala Ser Gly Ser Gly Phe Ser Ala Ser Leu Met Ser Trp His Arg Gln 370 375 380 Ala Pro Gly Lys Gln Arg Asp Leu Val Ala Ser Ile Thr Arg Asp Gly 385 390 395 400 Arg Ala Asn Tyr Val Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg 405 410 415 Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Ser Ser Leu Arg Ala 420 425 430 Glu Asp Thr Ala Val Tyr Tyr Cys His Ala Tyr Ser Phe Asp Tyr Pro 435 440 445 Ile Arg Ser Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Lys Pro Gly 450 455 460 Gly Gly Gly Ser Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly 465 470 475 480 Arg Thr Phe Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr Thr Gly 485 490 495 Met Leu Glu Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn Lys 500 505 510 Pro Phe Lys Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu 515 520 525 Glu Gly Val Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile 530 535 540 Ser Pro Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile Ile Pro 545 550 555 560 Pro His Ala Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu Glu Gly 565 570 575 Gly Arg Met Ala Leu Ile Val Leu Gly Gly Val Ala Gly Leu Leu Leu 580 585 590 Phe Ile Gly Leu Gly Ile Phe Phe Ala Leu Tyr Leu Leu Arg Arg Asp 595 600 605 Gln Arg Leu Pro Pro Asp Ala His Lys Pro Pro Gly Gly Gly Ser Phe 610 615 620 Arg Thr Pro Ile Gln Glu Glu Gln Ala Asp Ala His Ser Thr Leu Ala 625 630 635 640 Lys Ile <210> 45 <211> 642 <212> PRT <213> Artificial Sequence <220> <223> Made in Lab - anti-CD33 VHH DARIC.OX40 fusion protein <400> 45 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Gly Ser Ile Leu Trp His Glu Met Trp His Glu 20 25 30 Gly Leu Glu Glu Ala Ser Arg Leu Tyr Phe Gly Glu Arg Asn Val Lys 35 40 45 Gly Met Phe Glu Val Leu Glu Pro Leu His Ala Met Met Glu Arg Gly 50 55 60 Pro Gln Thr Leu Lys Glu Thr Ser Phe Asn Gln Ala Tyr Gly Arg Asp 65 70 75 80 Leu Met Glu Ala Gln Glu Trp Cys Arg Lys Tyr Met Lys Ser Gly Asn 85 90 95 Val Lys Asp Leu Leu Gln Ala Trp Asp Leu Tyr Tyr His Val Phe Arg 100 105 110 Arg Ile Ser Lys Ala Ser Ala Gly Thr Gly Ser Asp Ile Tyr Ile Trp 115 120 125 Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile 130 135 140 Thr Met His Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln 145 150 155 160 Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser 165 170 175 Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys 180 185 190 Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln 195 200 205 Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu 210 215 220 Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg 225 230 235 240 Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met 245 250 255 Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly 260 265 270 Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp 275 280 285 Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Ser Gly Ser 290 295 300 Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu 305 310 315 320 Asn Pro Gly Pro Ser Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu 325 330 335 Leu Leu Trp Val Pro Gly Ser Thr Gly Glu Val Gln Leu Val Glu Ser 340 345 350 Gly Gly Gly Glu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala 355 360 365 Ala Ser Gly Phe Thr Phe Ser Ser Tyr Ala Met Gly Trp Phe Arg Gln 370 375 380 Ala Pro Gly Lys Gly Arg Glu Trp Val Ala Ala Ile Thr Thr Ser Gly 385 390 395 400 Asp Thr Thr Tyr Tyr Ala Glu Ser Val Lys Gly Arg Phe Thr Ile Ser 405 410 415 Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Ser Ser Leu Arg 420 425 430 Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala His Arg Gly Gly Gly 435 440 445 Val Ile Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Lys Pro Gly 450 455 460 Gly Gly Gly Ser Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly 465 470 475 480 Arg Thr Phe Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr Thr Gly 485 490 495 Met Leu Glu Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn Lys 500 505 510 Pro Phe Lys Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu 515 520 525 Glu Gly Val Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile 530 535 540 Ser Pro Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile Ile Pro 545 550 555 560 Pro His Ala Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu Glu Gly 565 570 575 Gly Arg Met Ala Leu Ile Val Leu Gly Gly Val Ala Gly Leu Leu Leu 580 585 590 Phe Ile Gly Leu Gly Ile Phe Phe Ala Leu Tyr Leu Leu Arg Arg Asp 595 600 605 Gln Arg Leu Pro Pro Asp Ala His Lys Pro Pro Gly Gly Gly Ser Phe 610 615 620 Arg Thr Pro Ile Gln Glu Glu Gln Ala Asp Ala His Ser Thr Leu Ala 625 630 635 640 Lys Ile <210> 46 <211> 645 <212> PRT <213> Artificial Sequence <220> <223> Made in Lab - anti-CD33 VHH DARIC.OX40 fusion protein <400> 46 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Gly Ser Ile Leu Trp His Glu Met Trp His Glu 20 25 30 Gly Leu Glu Glu Ala Ser Arg Leu Tyr Phe Gly Glu Arg Asn Val Lys 35 40 45 Gly Met Phe Glu Val Leu Glu Pro Leu His Ala Met Met Glu Arg Gly 50 55 60 Pro Gln Thr Leu Lys Glu Thr Ser Phe Asn Gln Ala Tyr Gly Arg Asp 65 70 75 80 Leu Met Glu Ala Gln Glu Trp Cys Arg Lys Tyr Met Lys Ser Gly Asn 85 90 95 Val Lys Asp Leu Leu Gln Ala Trp Asp Leu Tyr Tyr His Val Phe Arg 100 105 110 Arg Ile Ser Lys Ala Ser Ala Gly Thr Gly Ser Asp Ile Tyr Ile Trp 115 120 125 Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile 130 135 140 Thr Met His Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln 145 150 155 160 Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser 165 170 175 Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys 180 185 190 Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln 195 200 205 Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu 210 215 220 Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg 225 230 235 240 Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met 245 250 255 Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly 260 265 270 Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp 275 280 285 Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Ser Gly Ser 290 295 300 Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu 305 310 315 320 Asn Pro Gly Pro Ser Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu 325 330 335 Leu Leu Trp Val Pro Gly Ser Thr Gly Glu Val Gln Leu Val Glu Ser 340 345 350 Gly Gly Gly Glu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala 355 360 365 Ala Ser Gly Ser Ile Phe Ser Ile Ser Ile Met Gly Trp Tyr Arg Gln 370 375 380 Ala Pro Gly Lys Glu Arg Glu Leu Val Ala Ser Thr Thr Ser Ser Gly 385 390 395 400 Thr Thr Asn Tyr Val Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg 405 410 415 Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Ser Ser Leu Arg Ala 420 425 430 Glu Asp Thr Ala Val Tyr Tyr Cys His Ala Tyr Ile Ala Thr Thr Thr 435 440 445 Asp Arg Gly Tyr Arg Gly Tyr Trp Gly Gln Gly Thr Leu Val Thr Val 450 455 460 Lys Pro Gly Gly Gly Gly Ser Gly Val Gln Val Glu Thr Ile Ser Pro 465 470 475 480 Gly Asp Gly Arg Thr Phe Pro Lys Arg Gly Gln Thr Cys Val Val His 485 490 495 Tyr Thr Gly Met Leu Glu Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp 500 505 510 Arg Asn Lys Pro Phe Lys Phe Met Leu Gly Lys Gln Glu Val Ile Arg 515 520 525 Gly Trp Glu Glu Gly Val Ala Gln Met Ser Val Gly Gln Arg Ala Lys 530 535 540 Leu Thr Ile Ser Pro Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly 545 550 555 560 Ile Ile Pro His Ala Thr Leu Val Phe Asp Val Glu Leu Leu Lys 565 570 575 Leu Glu Gly Gly Arg Met Ala Leu Ile Val Leu Gly Gly Val Ala Gly 580 585 590 Leu Leu Leu Phe Ile Gly Leu Gly Ile Phe Phe Ala Leu Tyr Leu Leu 595 600 605 Arg Arg Asp Gln Arg Leu Pro Pro Asp Ala His Lys Pro Pro Gly Gly 610 615 620 Gly Ser Phe Arg Thr Pro Ile Gln Glu Glu Gln Ala Asp Ala His Ser 625 630 635 640 Thr Leu Ala Lys Ile 645 <210> 47 <211> 648 <212> PRT <213> Artificial Sequence <220> <223> Made in Lab - anti-CD33 VHH DARIC.OX40 fusion protein <400> 47 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Gly Ser Ile Leu Trp His Glu Met Trp His Glu 20 25 30 Gly Leu Glu Glu Ala Ser Arg Leu Tyr Phe Gly Glu Arg Asn Val Lys 35 40 45 Gly Met Phe Glu Val Leu Glu Pro Leu His Ala Met Met Glu Arg Gly 50 55 60 Pro Gln Thr Leu Lys Glu Thr Ser Phe Asn Gln Ala Tyr Gly Arg Asp 65 70 75 80 Leu Met Glu Ala Gln Glu Trp Cys Arg Lys Tyr Met Lys Ser Gly Asn 85 90 95 Val Lys Asp Leu Leu Gln Ala Trp Asp Leu Tyr Tyr His Val Phe Arg 100 105 110 Arg Ile Ser Lys Ala Ser Ala Gly Thr Gly Ser Asp Ile Tyr Ile Trp 115 120 125 Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile 130 135 140 Thr Met His Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln 145 150 155 160 Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser 165 170 175 Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys 180 185 190 Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln 195 200 205 Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu 210 215 220 Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg 225 230 235 240 Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met 245 250 255 Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly 260 265 270 Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp 275 280 285 Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Ser Gly Ser 290 295 300 Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu 305 310 315 320 Asn Pro Gly Pro Ser Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu 325 330 335 Leu Leu Trp Val Pro Gly Ser Thr Gly Gln Val Gln Leu Val Gln Ser 340 345 350 Gly Gly Gly Leu Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Ala 355 360 365 Ala Ser Gly Ser Ile Ser Ser Tyr Asn Val Val Gly Trp Tyr Arg Gln 370 375 380 Leu Ser Gly Asn Glu Arg Gly Gly Arg Thr Met Val Ala Gln Ile Asn 385 390 395 400 Ala Tyr Gly Asp Thr Asn Tyr Ala Asn Ala Val Val Gly Arg Phe Thr 405 410 415 Ile Ser Arg Asp Asp Ala Lys Asn Thr Val Tyr Leu His Met Ser Asn 420 425 430 Leu Lys Pro Glu Asp Thr Gly Val Tyr Tyr Cys Asn Gly Gln Arg Met 435 440 445 Leu Glu Asn Tyr Thr Tyr Arg Asp Gln Ser Trp Gly Gln Gly Thr Gln 450 455 460 Val Thr Val Lys Pro Gly Gly Gly Gly Ser Gly Val Gln Val Glu Thr 465 470 475 480 Ile Ser Pro Gly Asp Gly Arg Thr Phe Pro Lys Arg Gly Gln Thr Cys 485 490 495 Val Val His Tyr Thr Gly Met Leu Glu Asp Gly Lys Lys Phe Asp Ser 500 505 510 Ser Arg Asp Arg Asn Lys Pro Phe Lys Phe Met Leu Gly Lys Gln Glu 515 520 525 Val Ile Arg Gly Trp Glu Glu Gly Val Ala Gln Met Ser Val Gly Gln 530 535 540 Arg Ala Lys Leu Thr Ile Ser Pro Asp Tyr Ala Tyr Gly Ala Thr Gly 545 550 555 560 His Pro Gly Ile Ile Pro His Ala Thr Leu Val Phe Asp Val Glu 565 570 575 Leu Leu Lys Leu Glu Gly Gly Arg Met Ala Leu Ile Val Leu Gly Gly 580 585 590 Val Ala Gly Leu Leu Leu Leu Phe Ile Gly Leu Gly Ile Phe Phe Ala Leu 595 600 605 Tyr Leu Leu Arg Arg Asp Gln Arg Leu Pro Pro Asp Ala His Lys Pro 610 615 620 Pro Gly Gly Gly Ser Phe Arg Thr Pro Ile Gln Glu Glu Gln Ala Asp 625 630 635 640 Ala His Ser Thr Leu Ala Lys Ile 645 <210> 48 <211> 640 <212> PRT <213> Artificial Sequence <220> <223> Made in Lab - anti-CD33 VHH DARIC.OX40 fusion protein <400> 48 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Gly Ser Ile Leu Trp His Glu Met Trp His Glu 20 25 30 Gly Leu Glu Glu Ala Ser Arg Leu Tyr Phe Gly Glu Arg Asn Val Lys 35 40 45 Gly Met Phe Glu Val Leu Glu Pro Leu His Ala Met Met Glu Arg Gly 50 55 60 Pro Gln Thr Leu Lys Glu Thr Ser Phe Asn Gln Ala Tyr Gly Arg Asp 65 70 75 80 Leu Met Glu Ala Gln Glu Trp Cys Arg Lys Tyr Met Lys Ser Gly Asn 85 90 95 Val Lys Asp Leu Leu Gln Ala Trp Asp Leu Tyr Tyr His Val Phe Arg 100 105 110 Arg Ile Ser Lys Ala Ser Ala Gly Thr Gly Ser Asp Ile Tyr Ile Trp 115 120 125 Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile 130 135 140 Thr Met His Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln 145 150 155 160 Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser 165 170 175 Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys 180 185 190 Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln 195 200 205 Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu 210 215 220 Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg 225 230 235 240 Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met 245 250 255 Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly 260 265 270 Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp 275 280 285 Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Ser Gly Ser 290 295 300 Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu 305 310 315 320 Asn Pro Gly Pro Ser Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu 325 330 335 Leu Leu Trp Val Pro Gly Ser Thr Gly Gln Val Gln Leu Val Gln Ser 340 345 350 Gly Gly Gly Leu Val Gln Ala Gly Gly Ser Leu Thr Leu Ser Cys Ala 355 360 365 Ala Ser Arg Ser Ser Gly Ile Asp Val Met Gly Trp Tyr Arg Gln Ala 370 375 380 Pro Gly Lys Glu Arg Glu Leu Val Ala Glu Ile Ser Gly Val Gly Asp 385 390 395 400 Thr Asn Tyr Ala Ala Ser Leu Ala Asp Arg Phe Thr Val Ser Arg Asp 405 410 415 Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Lys Asn Leu Lys Pro Glu 420 425 430 Asp Thr Ala Val Tyr Tyr Cys Asn Ala His Ser Phe Leu Asp Leu Val 435 440 445 Gly Ala Trp Gly Gln Gly Thr Gln Val Thr Val Lys Pro Gly Gly Gly 450 455 460 Gly Ser Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr 465 470 475 480 Phe Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr Thr Gly Met Leu 485 490 495 Glu Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe 500 505 510 Lys Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu Glu Gly 515 520 525 Val Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile Ser Pro 530 535 540 Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile Ile Pro His 545 550 555 560 Ala Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu Glu Gly Gly Arg 565 570 575 Met Ala Leu Ile Val Leu Gly Gly Val Ala Gly Leu Leu Leu Phe Ile 580 585 590 Gly Leu Gly Ile Phe Phe Ala Leu Tyr Leu Leu Arg Arg Asp Gln Arg 595 600 605 Leu Pro Pro Asp Ala His Lys Pro Pro Gly Gly Gly Ser Phe Arg Thr 610 615 620 Pro Ile Gln Glu Glu Gln Ala Asp Ala His Ser Thr Leu Ala Lys Ile 625 630 635 640 <210> 49 <211> 644 <212> PRT <213> Artificial Sequence <220> <223> Made in Lab - anti-CD33 VHH DARIC.OX40 fusion protein <400> 49 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Gly Ser Ile Leu Trp His Glu Met Trp His Glu 20 25 30 Gly Leu Glu Glu Ala Ser Arg Leu Tyr Phe Gly Glu Arg Asn Val Lys 35 40 45 Gly Met Phe Glu Val Leu Glu Pro Leu His Ala Met Met Glu Arg Gly 50 55 60 Pro Gln Thr Leu Lys Glu Thr Ser Phe Asn Gln Ala Tyr Gly Arg Asp 65 70 75 80 Leu Met Glu Ala Gln Glu Trp Cys Arg Lys Tyr Met Lys Ser Gly Asn 85 90 95 Val Lys Asp Leu Leu Gln Ala Trp Asp Leu Tyr Tyr His Val Phe Arg 100 105 110 Arg Ile Ser Lys Ala Ser Ala Gly Thr Gly Ser Asp Ile Tyr Ile Trp 115 120 125 Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile 130 135 140 Thr Met His Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln 145 150 155 160 Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser 165 170 175 Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys 180 185 190 Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln 195 200 205 Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu 210 215 220 Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg 225 230 235 240 Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met 245 250 255 Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly 260 265 270 Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp 275 280 285 Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Ser Gly Ser 290 295 300 Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu 305 310 315 320 Asn Pro Gly Pro Ser Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu 325 330 335 Leu Leu Trp Val Pro Gly Ser Thr Gly Glu Val Gln Leu Val Gln Ser 340 345 350 Gly Gly Gly Ser Val Gln Val Gly Gly Ser Leu Arg Leu Ser Cys Ala 355 360 365 Ala Ser Gly Ser Thr Leu Asn Ile Asp His Ile Gly Trp Tyr Arg Gln 370 375 380 Ala Pro Gly Lys Glu Arg Glu Leu Val Gly Val Ile Ser Ser Gly Ala 385 390 395 400 Gly Pro Asn Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg 405 410 415 Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro 420 425 430 Glu Asp Thr Ala Val Tyr Asn Cys Asn Ala Trp Ile Asp Tyr Gly Ser 435 440 445 Gly Leu Pro Gln Asn Tyr Trp Gly Gly Gly Thr Gln Val Thr Val Lys 450 455 460 Pro Gly Gly Gly Gly Ser Gly Val Gln Val Glu Thr Ile Ser Pro Gly 465 470 475 480 Asp Gly Arg Thr Phe Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr 485 490 495 Thr Gly Met Leu Glu Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg 500 505 510 Asn Lys Pro Phe Lys Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly 515 520 525 Trp Glu Glu Gly Val Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu 530 535 540 Thr Ile Ser Pro Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile 545 550 555 560 Ile Pro Pro His Ala Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu 565 570 575 Glu Gly Gly Arg Met Ala Leu Ile Val Leu Gly Gly Val Ala Gly Leu 580 585 590 Leu Leu Phe Ile Gly Leu Gly Ile Phe Phe Ala Leu Tyr Leu Leu Arg 595 600 605 Arg Asp Gln Arg Leu Pro Pro Asp Ala His Lys Pro Pro Gly Gly Gly 610 615 620 Ser Phe Arg Thr Pro Ile Gln Glu Glu Gln Ala Asp Ala His Ser Thr 625 630 635 640 Leu Ala Lys Ile <210> 50 <211> 640 <212> PRT <213> Artificial Sequence <220> <223> Made in Lab - anti-CD33 VHH DARIC.OX40 fusion protein <400> 50 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Gly Ser Ile Leu Trp His Glu Met Trp His Glu 20 25 30 Gly Leu Glu Glu Ala Ser Arg Leu Tyr Phe Gly Glu Arg Asn Val Lys 35 40 45 Gly Met Phe Glu Val Leu Glu Pro Leu His Ala Met Met Glu Arg Gly 50 55 60 Pro Gln Thr Leu Lys Glu Thr Ser Phe Asn Gln Ala Tyr Gly Arg Asp 65 70 75 80 Leu Met Glu Ala Gln Glu Trp Cys Arg Lys Tyr Met Lys Ser Gly Asn 85 90 95 Val Lys Asp Leu Leu Gln Ala Trp Asp Leu Tyr Tyr His Val Phe Arg 100 105 110 Arg Ile Ser Lys Ala Ser Ala Gly Thr Gly Ser Asp Ile Tyr Ile Trp 115 120 125 Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile 130 135 140 Thr Met His Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln 145 150 155 160 Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser 165 170 175 Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys 180 185 190 Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln 195 200 205 Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu 210 215 220 Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg 225 230 235 240 Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met 245 250 255 Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly 260 265 270 Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp 275 280 285 Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Ser Gly Ser 290 295 300 Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu 305 310 315 320 Asn Pro Gly Pro Ser Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu 325 330 335 Leu Leu Trp Val Pro Gly Ser Thr Gly Glu Val Gln Leu Val Glu Ser 340 345 350 Gly Gly Gly Glu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala 355 360 365 Ala Ser Arg Ser Ser Gly Ile Asp Val Met Gly Trp Tyr Arg Gln Ala 370 375 380 Pro Gly Lys Glu Arg Glu Leu Val Ala Glu Ile Ser Gly Val Gly Asp 385 390 395 400 Thr Asn Tyr Ala Ala Ser Leu Ala Asp Arg Phe Thr Val Ser Arg Asp 405 410 415 Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu 420 425 430 Asp Thr Ala Val Tyr Tyr Cys Asn Ala His Ser Phe Leu Asp Leu Val 435 440 445 Gly Ala Trp Gly Gln Gly Thr Leu Val Thr Val Lys Pro Gly Gly Gly 450 455 460 Gly Ser Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr 465 470 475 480 Phe Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr Thr Gly Met Leu 485 490 495 Glu Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe 500 505 510 Lys Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu Glu Gly 515 520 525 Val Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile Ser Pro 530 535 540 Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile Ile Pro His 545 550 555 560 Ala Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu Glu Gly Gly Arg 565 570 575 Met Ala Leu Ile Val Leu Gly Gly Val Ala Gly Leu Leu Leu Phe Ile 580 585 590 Gly Leu Gly Ile Phe Phe Ala Leu Tyr Leu Leu Arg Arg Asp Gln Arg 595 600 605 Leu Pro Pro Asp Ala His Lys Pro Pro Gly Gly Gly Ser Phe Arg Thr 610 615 620 Pro Ile Gln Glu Glu Gln Ala Asp Ala His Ser Thr Leu Ala Lys Ile 625 630 635 640 <210> 51 <211> 644 <212> PRT <213> Artificial Sequence <220> <223> Made in Lab - anti-CD33 VHH DARIC.OX40 fusion protein <400> 51 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Gly Ser Ile Leu Trp His Glu Met Trp His Glu 20 25 30 Gly Leu Glu Glu Ala Ser Arg Leu Tyr Phe Gly Glu Arg Asn Val Lys 35 40 45 Gly Met Phe Glu Val Leu Glu Pro Leu His Ala Met Met Glu Arg Gly 50 55 60 Pro Gln Thr Leu Lys Glu Thr Ser Phe Asn Gln Ala Tyr Gly Arg Asp 65 70 75 80 Leu Met Glu Ala Gln Glu Trp Cys Arg Lys Tyr Met Lys Ser Gly Asn 85 90 95 Val Lys Asp Leu Leu Gln Ala Trp Asp Leu Tyr Tyr His Val Phe Arg 100 105 110 Arg Ile Ser Lys Ala Ser Ala Gly Thr Gly Ser Asp Ile Tyr Ile Trp 115 120 125 Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile 130 135 140 Thr Met His Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln 145 150 155 160 Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser 165 170 175 Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys 180 185 190 Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln 195 200 205 Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu 210 215 220 Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg 225 230 235 240 Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met 245 250 255 Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly 260 265 270 Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp 275 280 285 Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Ser Gly Ser 290 295 300 Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu 305 310 315 320 Asn Pro Gly Pro Ser Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu 325 330 335 Leu Leu Trp Val Pro Gly Ser Thr Gly Glu Val Gln Leu Val Glu Ser 340 345 350 Gly Gly Gly Glu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala 355 360 365 Ala Ser Gly Ser Thr Leu Asn Ile Asp His Ile Gly Trp Tyr Arg Gln 370 375 380 Ala Pro Gly Lys Glu Arg Glu Leu Val Gly Val Ile Ser Ser Gly Ala 385 390 395 400 Gly Pro Asn Tyr Ala Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg 405 410 415 Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Ser Ser Leu Arg Ala 420 425 430 Glu Asp Thr Ala Val Tyr Tyr Cys Asn Ala Trp Ile Asp Tyr Gly Ser 435 440 445 Gly Leu Pro Gln Asn Tyr Trp Gly Gly Gly Thr Leu Val Thr Val Lys 450 455 460 Pro Gly Gly Gly Gly Ser Gly Val Gln Val Glu Thr Ile Ser Pro Gly 465 470 475 480 Asp Gly Arg Thr Phe Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr 485 490 495 Thr Gly Met Leu Glu Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg 500 505 510 Asn Lys Pro Phe Lys Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly 515 520 525 Trp Glu Glu Gly Val Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu 530 535 540 Thr Ile Ser Pro Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile 545 550 555 560 Ile Pro Pro His Ala Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu 565 570 575 Glu Gly Gly Arg Met Ala Leu Ile Val Leu Gly Gly Val Ala Gly Leu 580 585 590 Leu Leu Phe Ile Gly Leu Gly Ile Phe Phe Ala Leu Tyr Leu Leu Arg 595 600 605 Arg Asp Gln Arg Leu Pro Pro Asp Ala His Lys Pro Pro Gly Gly Gly 610 615 620 Ser Phe Arg Thr Pro Ile Gln Glu Glu Gln Ala Asp Ala His Ser Thr 625 630 635 640 Leu Ala Lys Ile <210> 52 <211> 781 <212> PRT <213> Artificial Sequence <220> <223> Made in Lab - anti-CD33 VHH DARIC.TNFR2 fusion protein <400> 52 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Gly Ser Ile Leu Trp His Glu Met Trp His Glu 20 25 30 Gly Leu Glu Glu Ala Ser Arg Leu Tyr Phe Gly Glu Arg Asn Val Lys 35 40 45 Gly Met Phe Glu Val Leu Glu Pro Leu His Ala Met Met Glu Arg Gly 50 55 60 Pro Gln Thr Leu Lys Glu Thr Ser Phe Asn Gln Ala Tyr Gly Arg Asp 65 70 75 80 Leu Met Glu Ala Gln Glu Trp Cys Arg Lys Tyr Met Lys Ser Gly Asn 85 90 95 Val Lys Asp Leu Leu Gln Ala Trp Asp Leu Tyr Tyr His Val Phe Arg 100 105 110 Arg Ile Ser Lys Ala Ser Ala Gly Thr Gly Ser Asp Ile Tyr Ile Trp 115 120 125 Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile 130 135 140 Thr Met His Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln 145 150 155 160 Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser 165 170 175 Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys 180 185 190 Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln 195 200 205 Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu 210 215 220 Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg 225 230 235 240 Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met 245 250 255 Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly 260 265 270 Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp 275 280 285 Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Ser Gly Ser 290 295 300 Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu 305 310 315 320 Asn Pro Gly Pro Ser Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu 325 330 335 Leu Leu Trp Val Pro Gly Ser Thr Gly Gin Val Thr Leu Arg Glu Ser 340 345 350 Gly Gly Gly Leu Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Lys 355 360 365 Gly Ser Gly Arg Ala Ile Asn Thr Tyr Ala Met Gly Trp Phe Arg Gln 370 375 380 Ala Pro Gly Lys Glu Arg Glu Phe Val Ala Ala Ile Ser Trp Asn Gly 385 390 395 400 Gly His Thr Arg Tyr Ala Asp Ser Val Gin Gly Arg Phe Ala Ile Ser 405 410 415 Arg Asp Asn Ala Asp Asn Thr Met Tyr Leu Gln Met Asn Ser Leu Lys 420 425 430 Pro Glu Asp Thr Ala Val Tyr His Cys Ala Ala Tyr Ser Asp Tyr His 435 440 445 Arg Ile Ala Thr Met Glu Ala Asp Ala Asp Ser Trp Gly Gln Gly Thr 450 455 460 Gln Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Val Gln Val Glu 465 470 475 480 Thr Ile Ser Pro Gly Asp Gly Arg Thr Phe Pro Lys Arg Gly Gln Thr 485 490 495 Cys Val Val His Tyr Thr Gly Met Leu Glu Asp Gly Lys Lys Phe Asp 500 505 510 Ser Ser Arg Asp Arg Asn Lys Pro Phe Lys Phe Met Leu Gly Lys Gln 515 520 525 Glu Val Ile Arg Gly Trp Glu Glu Gly Val Ala Gln Met Ser Val Gly 530 535 540 Gln Arg Ala Lys Leu Thr Ile Ser Pro Asp Tyr Ala Tyr Gly Ala Thr 545 550 555 560 Gly His Pro Gly Ile Ile Pro His Ala Thr Leu Val Phe Asp Val 565 570 575 Glu Leu Leu Lys Leu Glu Gly Gly Arg Met Ala Leu Ile Val Leu Gly 580 585 590 Gly Val Ala Gly Leu Leu Leu Phe Ile Gly Leu Gly Ile Phe Phe Lys 595 600 605 Lys Lys Pro Leu Cys Leu Gln Arg Glu Ala Lys Val Pro His Leu Pro 610 615 620 Ala Asp Lys Ala Arg Gly Thr Gln Gly Pro Glu Gln Gln His Leu Leu 625 630 635 640 Ile Thr Ala Pro Ser Ser Ser Ser Ser Ser Ser Leu Glu Ser Ser Ala Ser 645 650 655 Ala Leu Asp Arg Arg Ala Pro Thr Arg Asn Gln Pro Gln Ala Pro Gly 660 665 670 Val Glu Ala Ser Gly Ala Gly Glu Ala Arg Ala Ser Thr Gly Ser Ser 675 680 685 Asp Ser Ser Pro Gly Gly His Gly Thr Gln Val Asn Val Thr Cys Ile 690 695 700 Val Asn Val Cys Ser Ser Ser Ser Asp His Ser Ser Gln Cys Ser Ser Gln 705 710 715 720 Ala Ser Ser Thr Met Gly Asp Thr Asp Ser Ser Pro Ser Glu Ser Pro 725 730 735 Lys Asp Glu Gln Val Pro Phe Ser Lys Glu Glu Cys Ala Phe Arg Ser 740 745 750 Gln Leu Glu Thr Pro Glu Thr Leu Leu Gly Ser Thr Glu Glu Lys Pro 755 760 765 Leu Pro Leu Gly Val Pro Asp Ala Gly Met Lys Pro Ser 770 775 780 <210> 53 <211> 776 <212> PRT <213> Artificial Sequence <220> <223> Made in Lab - anti-CD33 VHH DARIC.TNFR2 fusion protein <400> 53 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Gly Ser Ile Leu Trp His Glu Met Trp His Glu 20 25 30 Gly Leu Glu Glu Ala Ser Arg Leu Tyr Phe Gly Glu Arg Asn Val Lys 35 40 45 Gly Met Phe Glu Val Leu Glu Pro Leu His Ala Met Met Glu Arg Gly 50 55 60 Pro Gln Thr Leu Lys Glu Thr Ser Phe Asn Gln Ala Tyr Gly Arg Asp 65 70 75 80 Leu Met Glu Ala Gln Glu Trp Cys Arg Lys Tyr Met Lys Ser Gly Asn 85 90 95 Val Lys Asp Leu Leu Gln Ala Trp Asp Leu Tyr Tyr His Val Phe Arg 100 105 110 Arg Ile Ser Lys Ala Ser Ala Gly Thr Gly Ser Asp Ile Tyr Ile Trp 115 120 125 Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile 130 135 140 Thr Met His Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln 145 150 155 160 Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser 165 170 175 Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys 180 185 190 Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln 195 200 205 Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu 210 215 220 Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg 225 230 235 240 Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met 245 250 255 Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly 260 265 270 Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp 275 280 285 Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Ser Gly Ser 290 295 300 Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu 305 310 315 320 Asn Pro Gly Pro Ser Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu 325 330 335 Leu Leu Trp Val Pro Gly Ser Thr Gly Glu Val Gln Leu Val Glu Ser 340 345 350 Gly Gly Gly Glu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala 355 360 365 Ala Ser Gly Arg Thr Phe Ser Gly Tyr Ile Met Gly Trp Phe Arg Gln 370 375 380 Ala Pro Gly Lys Glu Arg Glu Leu Val Ala Arg Ile Ser Gly Asn Asn 385 390 395 400 Leu Ser Thr Glu Tyr Ala Glu Ser Val Lys Gly Arg Phe Thr Ile Ser 405 410 415 Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Ser Ser Leu Arg 420 425 430 Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala Glu Tyr Asp Tyr Ser 435 440 445 Ser Gly Asp Phe Val Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Lys 450 455 460 Pro Gly Gly Gly Gly Ser Gly Val Gln Val Glu Thr Ile Ser Pro Gly 465 470 475 480 Asp Gly Arg Thr Phe Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr 485 490 495 Thr Gly Met Leu Glu Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg 500 505 510 Asn Lys Pro Phe Lys Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly 515 520 525 Trp Glu Glu Gly Val Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu 530 535 540 Thr Ile Ser Pro Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile 545 550 555 560 Ile Pro Pro His Ala Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu 565 570 575 Glu Gly Gly Arg Met Ala Leu Ile Val Leu Gly Gly Val Ala Gly Leu 580 585 590 Leu Leu Phe Ile Gly Leu Gly Ile Phe Phe Lys Lys Lys Lys Pro Leu Cys 595 600 605 Leu Gln Arg Glu Ala Lys Val Pro His Leu Pro Ala Asp Lys Ala Arg 610 615 620 Gly Thr Gln Gly Pro Glu Gln Gln His Leu Leu Ile Thr Ala Pro Ser 625 630 635 640 Ser Ser Ser Ser Ser Leu Glu Ser Ser Ala Ser Ala Leu Asp Arg Arg 645 650 655 Ala Pro Thr Arg Asn Gln Pro Gln Ala Pro Gly Val Glu Ala Ser Gly 660 665 670 Ala Gly Glu Ala Arg Ala Ser Thr Gly Ser Ser Asp Ser Ser Pro Gly 675 680 685 Gly His Gly Thr Gln Val Asn Val Thr Cys Ile Val Asn Val Cys Ser 690 695 700 Ser Ser Asp His Ser Ser Gln Cys Ser Ser Gln Ala Ser Ser Thr Met 705 710 715 720 Gly Asp Thr Asp Ser Ser Pro Ser Glu Ser Pro Lys Asp Glu Gln Val 725 730 735 Pro Phe Ser Lys Glu Glu Cys Ala Phe Arg Ser Gln Leu Glu Thr Pro 740 745 750 Glu Thr Leu Leu Gly Ser Thr Glu Glu Lys Pro Leu Pro Leu Gly Val 755 760 765 Pro Asp Ala Gly Met Lys Pro Ser 770 775 <210> 54 <211> 774 <212> PRT <213> Artificial Sequence <220> <223> Made in Lab - anti-CD33 VHH DARIC.TNFR2 fusion protein <400> 54 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Gly Ser Ile Leu Trp His Glu Met Trp His Glu 20 25 30 Gly Leu Glu Glu Ala Ser Arg Leu Tyr Phe Gly Glu Arg Asn Val Lys 35 40 45 Gly Met Phe Glu Val Leu Glu Pro Leu His Ala Met Met Glu Arg Gly 50 55 60 Pro Gln Thr Leu Lys Glu Thr Ser Phe Asn Gln Ala Tyr Gly Arg Asp 65 70 75 80 Leu Met Glu Ala Gln Glu Trp Cys Arg Lys Tyr Met Lys Ser Gly Asn 85 90 95 Val Lys Asp Leu Leu Gln Ala Trp Asp Leu Tyr Tyr His Val Phe Arg 100 105 110 Arg Ile Ser Lys Ala Ser Ala Gly Thr Gly Ser Asp Ile Tyr Ile Trp 115 120 125 Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile 130 135 140 Thr Met His Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln 145 150 155 160 Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser 165 170 175 Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys 180 185 190 Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln 195 200 205 Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu 210 215 220 Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg 225 230 235 240 Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met 245 250 255 Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly 260 265 270 Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp 275 280 285 Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Ser Gly Ser 290 295 300 Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu 305 310 315 320 Asn Pro Gly Pro Ser Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu 325 330 335 Leu Leu Trp Val Pro Gly Ser Thr Gly Glu Val Gln Leu Val Glu Ser 340 345 350 Gly Gly Gly Glu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala 355 360 365 Ala Ser Gly Ser Gly Phe Ser Ala Ser Leu Met Ser Trp His Arg Gln 370 375 380 Ala Pro Gly Lys Gln Arg Asp Leu Val Ala Ser Ile Thr Arg Asp Gly 385 390 395 400 Arg Ala Asn Tyr Val Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg 405 410 415 Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Ser Ser Leu Arg Ala 420 425 430 Glu Asp Thr Ala Val Tyr Tyr Cys His Ala Tyr Ser Phe Asp Tyr Pro 435 440 445 Ile Arg Ser Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Lys Pro Gly 450 455 460 Gly Gly Gly Ser Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly 465 470 475 480 Arg Thr Phe Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr Thr Gly 485 490 495 Met Leu Glu Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn Lys 500 505 510 Pro Phe Lys Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu 515 520 525 Glu Gly Val Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile 530 535 540 Ser Pro Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile Ile Pro 545 550 555 560 Pro His Ala Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu Glu Gly 565 570 575 Gly Arg Met Ala Leu Ile Val Leu Gly Gly Val Ala Gly Leu Leu Leu 580 585 590 Phe Ile Gly Leu Gly Ile Phe Phe Lys Lys Lys Pro Leu Cys Leu Gln 595 600 605 Arg Glu Ala Lys Val Pro His Leu Pro Ala Asp Lys Ala Arg Gly Thr 610 615 620 Gln Gly Pro Glu Gln Gln His Leu Leu Ile Thr Ala Pro Ser Ser Ser Ser 625 630 635 640 Ser Ser Ser Leu Glu Ser Ser Ser Ala Ser Ala Leu Asp Arg Arg Ala Pro 645 650 655 Thr Arg Asn Gln Pro Gln Ala Pro Gly Val Glu Ala Ser Gly Ala Gly 660 665 670 Glu Ala Arg Ala Ser Thr Gly Ser Ser Asp Ser Ser Pro Gly Gly His 675 680 685 Gly Thr Gln Val Asn Val Thr Cys Ile Val Asn Val Cys Ser Ser Ser Ser 690 695 700 Asp His Ser Ser Gln Cys Ser Ser Gln Ala Ser Ser Thr Met Gly Asp 705 710 715 720 Thr Asp Ser Ser Pro Ser Glu Ser Pro Lys Asp Glu Gln Val Pro Phe 725 730 735 Ser Lys Glu Glu Cys Ala Phe Arg Ser Gln Leu Glu Thr Pro Glu Thr 740 745 750 Leu Leu Gly Ser Thr Glu Glu Lys Pro Leu Pro Leu Gly Val Pro Asp 755 760 765 Ala Gly Met Lys Pro Ser 770 <210> 55 <211> 774 <212> PRT <213> Artificial Sequence <220> <223> Made in Lab - anti-CD33 VHH DARIC.TNFR2 fusion protein <400> 55 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Gly Ser Ile Leu Trp His Glu Met Trp His Glu 20 25 30 Gly Leu Glu Glu Ala Ser Arg Leu Tyr Phe Gly Glu Arg Asn Val Lys 35 40 45 Gly Met Phe Glu Val Leu Glu Pro Leu His Ala Met Met Glu Arg Gly 50 55 60 Pro Gln Thr Leu Lys Glu Thr Ser Phe Asn Gln Ala Tyr Gly Arg Asp 65 70 75 80 Leu Met Glu Ala Gln Glu Trp Cys Arg Lys Tyr Met Lys Ser Gly Asn 85 90 95 Val Lys Asp Leu Leu Gln Ala Trp Asp Leu Tyr Tyr His Val Phe Arg 100 105 110 Arg Ile Ser Lys Ala Ser Ala Gly Thr Gly Ser Asp Ile Tyr Ile Trp 115 120 125 Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile 130 135 140 Thr Met His Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln 145 150 155 160 Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser 165 170 175 Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys 180 185 190 Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln 195 200 205 Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu 210 215 220 Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg 225 230 235 240 Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met 245 250 255 Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly 260 265 270 Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp 275 280 285 Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Ser Gly Ser 290 295 300 Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu 305 310 315 320 Asn Pro Gly Pro Ser Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu 325 330 335 Leu Leu Trp Val Pro Gly Ser Thr Gly Glu Val Gln Leu Val Glu Ser 340 345 350 Gly Gly Gly Glu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala 355 360 365 Ala Ser Gly Phe Thr Phe Ser Ser Tyr Ala Met Gly Trp Phe Arg Gln 370 375 380 Ala Pro Gly Lys Gly Arg Glu Trp Val Ala Ala Ile Thr Thr Ser Gly 385 390 395 400 Asp Thr Thr Tyr Tyr Ala Glu Ser Val Lys Gly Arg Phe Thr Ile Ser 405 410 415 Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Ser Ser Leu Arg 420 425 430 Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala His Arg Gly Gly Gly 435 440 445 Val Ile Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Lys Pro Gly 450 455 460 Gly Gly Gly Ser Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly 465 470 475 480 Arg Thr Phe Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr Thr Gly 485 490 495 Met Leu Glu Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn Lys 500 505 510 Pro Phe Lys Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu 515 520 525 Glu Gly Val Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile 530 535 540 Ser Pro Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile Ile Pro 545 550 555 560 Pro His Ala Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu Glu Gly 565 570 575 Gly Arg Met Ala Leu Ile Val Leu Gly Gly Val Ala Gly Leu Leu Leu 580 585 590 Phe Ile Gly Leu Gly Ile Phe Phe Lys Lys Lys Pro Leu Cys Leu Gln 595 600 605 Arg Glu Ala Lys Val Pro His Leu Pro Ala Asp Lys Ala Arg Gly Thr 610 615 620 Gln Gly Pro Glu Gln Gln His Leu Leu Ile Thr Ala Pro Ser Ser Ser Ser 625 630 635 640 Ser Ser Ser Leu Glu Ser Ser Ser Ala Ser Ala Leu Asp Arg Arg Ala Pro 645 650 655 Thr Arg Asn Gln Pro Gln Ala Pro Gly Val Glu Ala Ser Gly Ala Gly 660 665 670 Glu Ala Arg Ala Ser Thr Gly Ser Ser Asp Ser Ser Pro Gly Gly His 675 680 685 Gly Thr Gln Val Asn Val Thr Cys Ile Val Asn Val Cys Ser Ser Ser Ser 690 695 700 Asp His Ser Ser Gln Cys Ser Ser Gln Ala Ser Ser Thr Met Gly Asp 705 710 715 720 Thr Asp Ser Ser Pro Ser Glu Ser Pro Lys Asp Glu Gln Val Pro Phe 725 730 735 Ser Lys Glu Glu Cys Ala Phe Arg Ser Gln Leu Glu Thr Pro Glu Thr 740 745 750 Leu Leu Gly Ser Thr Glu Glu Lys Pro Leu Pro Leu Gly Val Pro Asp 755 760 765 Ala Gly Met Lys Pro Ser 770 <210> 56 <211> 777 <212> PRT <213> Artificial Sequence <220> <223> Made in Lab - anti-CD33 VHH DARIC.TNFR2 fusion protein <400> 56 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Gly Ser Ile Leu Trp His Glu Met Trp His Glu 20 25 30 Gly Leu Glu Glu Ala Ser Arg Leu Tyr Phe Gly Glu Arg Asn Val Lys 35 40 45 Gly Met Phe Glu Val Leu Glu Pro Leu His Ala Met Met Glu Arg Gly 50 55 60 Pro Gln Thr Leu Lys Glu Thr Ser Phe Asn Gln Ala Tyr Gly Arg Asp 65 70 75 80 Leu Met Glu Ala Gln Glu Trp Cys Arg Lys Tyr Met Lys Ser Gly Asn 85 90 95 Val Lys Asp Leu Leu Gln Ala Trp Asp Leu Tyr Tyr His Val Phe Arg 100 105 110 Arg Ile Ser Lys Ala Ser Ala Gly Thr Gly Ser Asp Ile Tyr Ile Trp 115 120 125 Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile 130 135 140 Thr Met His Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln 145 150 155 160 Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser 165 170 175 Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys 180 185 190 Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln 195 200 205 Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu 210 215 220 Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg 225 230 235 240 Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met 245 250 255 Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly 260 265 270 Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp 275 280 285 Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Ser Gly Ser 290 295 300 Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu 305 310 315 320 Asn Pro Gly Pro Ser Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu 325 330 335 Leu Leu Trp Val Pro Gly Ser Thr Gly Glu Val Gln Leu Val Glu Ser 340 345 350 Gly Gly Gly Glu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala 355 360 365 Ala Ser Gly Ser Ile Phe Ser Ile Ser Ile Met Gly Trp Tyr Arg Gln 370 375 380 Ala Pro Gly Lys Glu Arg Glu Leu Val Ala Ser Thr Thr Ser Ser Gly 385 390 395 400 Thr Thr Asn Tyr Val Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg 405 410 415 Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Ser Ser Leu Arg Ala 420 425 430 Glu Asp Thr Ala Val Tyr Tyr Cys His Ala Tyr Ile Ala Thr Thr Thr 435 440 445 Asp Arg Gly Tyr Arg Gly Tyr Trp Gly Gln Gly Thr Leu Val Thr Val 450 455 460 Lys Pro Gly Gly Gly Gly Ser Gly Val Gln Val Glu Thr Ile Ser Pro 465 470 475 480 Gly Asp Gly Arg Thr Phe Pro Lys Arg Gly Gln Thr Cys Val Val His 485 490 495 Tyr Thr Gly Met Leu Glu Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp 500 505 510 Arg Asn Lys Pro Phe Lys Phe Met Leu Gly Lys Gln Glu Val Ile Arg 515 520 525 Gly Trp Glu Glu Gly Val Ala Gln Met Ser Val Gly Gln Arg Ala Lys 530 535 540 Leu Thr Ile Ser Pro Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly 545 550 555 560 Ile Ile Pro His Ala Thr Leu Val Phe Asp Val Glu Leu Leu Lys 565 570 575 Leu Glu Gly Gly Arg Met Ala Leu Ile Val Leu Gly Gly Val Ala Gly 580 585 590 Leu Leu Leu Phe Ile Gly Leu Gly Ile Phe Phe Lys Lys Lys Pro Leu 595 600 605 Cys Leu Gln Arg Glu Ala Lys Val Pro His Leu Pro Ala Asp Lys Ala 610 615 620 Arg Gly Thr Gln Gly Pro Glu Gln Gln His Leu Leu Ile Thr Ala Pro 625 630 635 640 Ser Ser Ser Ser Ser Ser Leu Glu Ser Ser Ala Ser Ala Leu Asp Arg 645 650 655 Arg Ala Pro Thr Arg Asn Gln Pro Gln Ala Pro Gly Val Glu Ala Ser 660 665 670 Gly Ala Gly Glu Ala Arg Ala Ser Thr Gly Ser Ser Asp Ser Ser Pro 675 680 685 Gly Gly His Gly Thr Gln Val Asn Val Thr Cys Ile Val Asn Val Cys 690 695 700 Ser Ser Ser Asp His Ser Ser Gln Cys Ser Ser Gln Ala Ser Ser Thr 705 710 715 720 Met Gly Asp Thr Asp Ser Ser Pro Ser Glu Ser Pro Lys Asp Glu Gln 725 730 735 Val Pro Phe Ser Lys Glu Glu Cys Ala Phe Arg Ser Gln Leu Glu Thr 740 745 750 Pro Glu Thr Leu Leu Gly Ser Thr Glu Glu Lys Pro Leu Pro Leu Gly 755 760 765 Val Pro Asp Ala Gly Met Lys Pro Ser 770 775 <210> 57 <211> 780 <212> PRT <213> Artificial Sequence <220> <223> Made in Lab - anti-CD33 VHH DARIC.TNFR2 fusion protein <400> 57 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Gly Ser Ile Leu Trp His Glu Met Trp His Glu 20 25 30 Gly Leu Glu Glu Ala Ser Arg Leu Tyr Phe Gly Glu Arg Asn Val Lys 35 40 45 Gly Met Phe Glu Val Leu Glu Pro Leu His Ala Met Met Glu Arg Gly 50 55 60 Pro Gln Thr Leu Lys Glu Thr Ser Phe Asn Gln Ala Tyr Gly Arg Asp 65 70 75 80 Leu Met Glu Ala Gln Glu Trp Cys Arg Lys Tyr Met Lys Ser Gly Asn 85 90 95 Val Lys Asp Leu Leu Gln Ala Trp Asp Leu Tyr Tyr His Val Phe Arg 100 105 110 Arg Ile Ser Lys Ala Ser Ala Gly Thr Gly Ser Asp Ile Tyr Ile Trp 115 120 125 Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile 130 135 140 Thr Met His Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln 145 150 155 160 Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser 165 170 175 Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys 180 185 190 Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln 195 200 205 Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu 210 215 220 Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg 225 230 235 240 Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met 245 250 255 Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly 260 265 270 Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp 275 280 285 Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Ser Gly Ser 290 295 300 Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu 305 310 315 320 Asn Pro Gly Pro Ser Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu 325 330 335 Leu Leu Trp Val Pro Gly Ser Thr Gly Gln Val Gln Leu Val Gln Ser 340 345 350 Gly Gly Gly Leu Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Ala 355 360 365 Ala Ser Gly Ser Ile Ser Ser Tyr Asn Val Val Gly Trp Tyr Arg Gln 370 375 380 Leu Ser Gly Asn Glu Arg Gly Gly Arg Thr Met Val Ala Gln Ile Asn 385 390 395 400 Ala Tyr Gly Asp Thr Asn Tyr Ala Asn Ala Val Val Gly Arg Phe Thr 405 410 415 Ile Ser Arg Asp Asp Ala Lys Asn Thr Val Tyr Leu His Met Ser Asn 420 425 430 Leu Lys Pro Glu Asp Thr Gly Val Tyr Tyr Cys Asn Gly Gln Arg Met 435 440 445 Leu Glu Asn Tyr Thr Tyr Arg Asp Gln Ser Trp Gly Gln Gly Thr Gln 450 455 460 Val Thr Val Lys Pro Gly Gly Gly Gly Ser Gly Val Gln Val Glu Thr 465 470 475 480 Ile Ser Pro Gly Asp Gly Arg Thr Phe Pro Lys Arg Gly Gln Thr Cys 485 490 495 Val Val His Tyr Thr Gly Met Leu Glu Asp Gly Lys Lys Phe Asp Ser 500 505 510 Ser Arg Asp Arg Asn Lys Pro Phe Lys Phe Met Leu Gly Lys Gln Glu 515 520 525 Val Ile Arg Gly Trp Glu Glu Gly Val Ala Gln Met Ser Val Gly Gln 530 535 540 Arg Ala Lys Leu Thr Ile Ser Pro Asp Tyr Ala Tyr Gly Ala Thr Gly 545 550 555 560 His Pro Gly Ile Ile Pro His Ala Thr Leu Val Phe Asp Val Glu 565 570 575 Leu Leu Lys Leu Glu Gly Gly Arg Met Ala Leu Ile Val Leu Gly Gly 580 585 590 Val Ala Gly Leu Leu Leu Phe Ile Gly Leu Gly Ile Phe Phe Lys Lys 595 600 605 Lys Pro Leu Cys Leu Gln Arg Glu Ala Lys Val Pro His Leu Pro Ala 610 615 620 Asp Lys Ala Arg Gly Thr Gln Gly Pro Glu Gln Gln His Leu Leu Ile 625 630 635 640 Thr Ala Pro Ser Ser Ser Ser Ser Ser Ser Leu Glu Ser Ser Ala Ser Ala 645 650 655 Leu Asp Arg Arg Ala Pro Thr Arg Asn Gln Pro Gln Ala Pro Gly Val 660 665 670 Glu Ala Ser Gly Ala Gly Glu Ala Arg Ala Ser Thr Gly Ser Ser Asp 675 680 685 Ser Ser Pro Gly Gly His Gly Thr Gln Val Asn Val Thr Cys Ile Val 690 695 700 Asn Val Cys Ser Ser Ser Asp His Ser Ser Gln Cys Ser Ser Gln Ala 705 710 715 720 Ser Ser Thr Met Gly Asp Thr Asp Ser Ser Pro Ser Glu Ser Pro Lys 725 730 735 Asp Glu Gln Val Pro Phe Ser Lys Glu Glu Cys Ala Phe Arg Ser Gln 740 745 750 Leu Glu Thr Pro Glu Thr Leu Leu Gly Ser Thr Glu Glu Lys Pro Leu 755 760 765 Pro Leu Gly Val Pro Asp Ala Gly Met Lys Pro Ser 770 775 780 <210> 58 <211> 772 <212> PRT <213> Artificial Sequence <220> <223> Made in Lab - anti-CD33 VHH DARIC.TNFR2 fusion protein <400> 58 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Gly Ser Ile Leu Trp His Glu Met Trp His Glu 20 25 30 Gly Leu Glu Glu Ala Ser Arg Leu Tyr Phe Gly Glu Arg Asn Val Lys 35 40 45 Gly Met Phe Glu Val Leu Glu Pro Leu His Ala Met Met Glu Arg Gly 50 55 60 Pro Gln Thr Leu Lys Glu Thr Ser Phe Asn Gln Ala Tyr Gly Arg Asp 65 70 75 80 Leu Met Glu Ala Gln Glu Trp Cys Arg Lys Tyr Met Lys Ser Gly Asn 85 90 95 Val Lys Asp Leu Leu Gln Ala Trp Asp Leu Tyr Tyr His Val Phe Arg 100 105 110 Arg Ile Ser Lys Ala Ser Ala Gly Thr Gly Ser Asp Ile Tyr Ile Trp 115 120 125 Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile 130 135 140 Thr Met His Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln 145 150 155 160 Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser 165 170 175 Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys 180 185 190 Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln 195 200 205 Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu 210 215 220 Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg 225 230 235 240 Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met 245 250 255 Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly 260 265 270 Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp 275 280 285 Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Ser Gly Ser 290 295 300 Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu 305 310 315 320 Asn Pro Gly Pro Ser Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu 325 330 335 Leu Leu Trp Val Pro Gly Ser Thr Gly Gln Val Gln Leu Val Gln Ser 340 345 350 Gly Gly Gly Leu Val Gln Ala Gly Gly Ser Leu Thr Leu Ser Cys Ala 355 360 365 Ala Ser Arg Ser Ser Gly Ile Asp Val Met Gly Trp Tyr Arg Gln Ala 370 375 380 Pro Gly Lys Glu Arg Glu Leu Val Ala Glu Ile Ser Gly Val Gly Asp 385 390 395 400 Thr Asn Tyr Ala Ala Ser Leu Ala Asp Arg Phe Thr Val Ser Arg Asp 405 410 415 Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Lys Asn Leu Lys Pro Glu 420 425 430 Asp Thr Ala Val Tyr Tyr Cys Asn Ala His Ser Phe Leu Asp Leu Val 435 440 445 Gly Ala Trp Gly Gln Gly Thr Gln Val Thr Val Lys Pro Gly Gly Gly 450 455 460 Gly Ser Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr 465 470 475 480 Phe Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr Thr Gly Met Leu 485 490 495 Glu Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe 500 505 510 Lys Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu Glu Gly 515 520 525 Val Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile Ser Pro 530 535 540 Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile Ile Pro His 545 550 555 560 Ala Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu Glu Gly Gly Arg 565 570 575 Met Ala Leu Ile Val Leu Gly Gly Val Ala Gly Leu Leu Leu Phe Ile 580 585 590 Gly Leu Gly Ile Phe Phe Lys Lys Lys Pro Leu Cys Leu Gln Arg Glu 595 600 605 Ala Lys Val Pro His Leu Pro Ala Asp Lys Ala Arg Gly Thr Gln Gly 610 615 620 Pro Glu Gln Gln His Leu Leu Ile Thr Ala Pro Ser Ser Ser Ser Ser Ser 625 630 635 640 Ser Leu Glu Ser Ser Ala Ser Ala Leu Asp Arg Arg Ala Pro Thr Arg 645 650 655 Asn Gln Pro Gln Ala Pro Gly Val Glu Ala Ser Gly Ala Gly Glu Ala 660 665 670 Arg Ala Ser Thr Gly Ser Ser Asp Ser Ser Pro Gly Gly His Gly Thr 675 680 685 Gln Val Asn Val Thr Cys Ile Val Asn Val Cys Ser Ser Ser Asp His 690 695 700 Ser Ser Gln Cys Ser Ser Gln Ala Ser Ser Thr Met Gly Asp Thr Asp 705 710 715 720 Ser Ser Pro Ser Glu Ser Pro Lys Asp Glu Gln Val Pro Phe Ser Lys 725 730 735 Glu Glu Cys Ala Phe Arg Ser Gln Leu Glu Thr Pro Glu Thr Leu Leu 740 745 750 Gly Ser Thr Glu Glu Lys Pro Leu Pro Leu Gly Val Pro Asp Ala Gly 755 760 765 Met Lys Pro Ser 770 <210> 59 <211> 776 <212> PRT <213> Artificial Sequence <220> <223> Made in Lab - anti-CD33 VHH DARIC.TNFR2 fusion protein <400> 59 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Gly Ser Ile Leu Trp His Glu Met Trp His Glu 20 25 30 Gly Leu Glu Glu Ala Ser Arg Leu Tyr Phe Gly Glu Arg Asn Val Lys 35 40 45 Gly Met Phe Glu Val Leu Glu Pro Leu His Ala Met Met Glu Arg Gly 50 55 60 Pro Gln Thr Leu Lys Glu Thr Ser Phe Asn Gln Ala Tyr Gly Arg Asp 65 70 75 80 Leu Met Glu Ala Gln Glu Trp Cys Arg Lys Tyr Met Lys Ser Gly Asn 85 90 95 Val Lys Asp Leu Leu Gln Ala Trp Asp Leu Tyr Tyr His Val Phe Arg 100 105 110 Arg Ile Ser Lys Ala Ser Ala Gly Thr Gly Ser Asp Ile Tyr Ile Trp 115 120 125 Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile 130 135 140 Thr Met His Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln 145 150 155 160 Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser 165 170 175 Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys 180 185 190 Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln 195 200 205 Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu 210 215 220 Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg 225 230 235 240 Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met 245 250 255 Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly 260 265 270 Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp 275 280 285 Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Ser Gly Ser 290 295 300 Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu 305 310 315 320 Asn Pro Gly Pro Ser Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu 325 330 335 Leu Leu Trp Val Pro Gly Ser Thr Gly Glu Val Gln Leu Val Gln Ser 340 345 350 Gly Gly Gly Ser Val Gln Val Gly Gly Ser Leu Arg Leu Ser Cys Ala 355 360 365 Ala Ser Gly Ser Thr Leu Asn Ile Asp His Ile Gly Trp Tyr Arg Gln 370 375 380 Ala Pro Gly Lys Glu Arg Glu Leu Val Gly Val Ile Ser Ser Gly Ala 385 390 395 400 Gly Pro Asn Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg 405 410 415 Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro 420 425 430 Glu Asp Thr Ala Val Tyr Asn Cys Asn Ala Trp Ile Asp Tyr Gly Ser 435 440 445 Gly Leu Pro Gln Asn Tyr Trp Gly Gly Gly Thr Gln Val Thr Val Lys 450 455 460 Pro Gly Gly Gly Gly Ser Gly Val Gln Val Glu Thr Ile Ser Pro Gly 465 470 475 480 Asp Gly Arg Thr Phe Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr 485 490 495 Thr Gly Met Leu Glu Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg 500 505 510 Asn Lys Pro Phe Lys Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly 515 520 525 Trp Glu Glu Gly Val Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu 530 535 540 Thr Ile Ser Pro Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile 545 550 555 560 Ile Pro Pro His Ala Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu 565 570 575 Glu Gly Gly Arg Met Ala Leu Ile Val Leu Gly Gly Val Ala Gly Leu 580 585 590 Leu Leu Phe Ile Gly Leu Gly Ile Phe Phe Lys Lys Lys Lys Pro Leu Cys 595 600 605 Leu Gln Arg Glu Ala Lys Val Pro His Leu Pro Ala Asp Lys Ala Arg 610 615 620 Gly Thr Gln Gly Pro Glu Gln Gln His Leu Leu Ile Thr Ala Pro Ser 625 630 635 640 Ser Ser Ser Ser Ser Leu Glu Ser Ser Ala Ser Ala Leu Asp Arg Arg 645 650 655 Ala Pro Thr Arg Asn Gln Pro Gln Ala Pro Gly Val Glu Ala Ser Gly 660 665 670 Ala Gly Glu Ala Arg Ala Ser Thr Gly Ser Ser Asp Ser Ser Pro Gly 675 680 685 Gly His Gly Thr Gln Val Asn Val Thr Cys Ile Val Asn Val Cys Ser 690 695 700 Ser Ser Asp His Ser Ser Gln Cys Ser Ser Gln Ala Ser Ser Thr Met 705 710 715 720 Gly Asp Thr Asp Ser Ser Pro Ser Glu Ser Pro Lys Asp Glu Gln Val 725 730 735 Pro Phe Ser Lys Glu Glu Cys Ala Phe Arg Ser Gln Leu Glu Thr Pro 740 745 750 Glu Thr Leu Leu Gly Ser Thr Glu Glu Lys Pro Leu Pro Leu Gly Val 755 760 765 Pro Asp Ala Gly Met Lys Pro Ser 770 775 <210> 60 <211> 772 <212> PRT <213> Artificial Sequence <220> <223> Made in Lab - anti-CD33 VHH DARIC.TNFR2 fusion protein <400> 60 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Gly Ser Ile Leu Trp His Glu Met Trp His Glu 20 25 30 Gly Leu Glu Glu Ala Ser Arg Leu Tyr Phe Gly Glu Arg Asn Val Lys 35 40 45 Gly Met Phe Glu Val Leu Glu Pro Leu His Ala Met Met Glu Arg Gly 50 55 60 Pro Gln Thr Leu Lys Glu Thr Ser Phe Asn Gln Ala Tyr Gly Arg Asp 65 70 75 80 Leu Met Glu Ala Gln Glu Trp Cys Arg Lys Tyr Met Lys Ser Gly Asn 85 90 95 Val Lys Asp Leu Leu Gln Ala Trp Asp Leu Tyr Tyr His Val Phe Arg 100 105 110 Arg Ile Ser Lys Ala Ser Ala Gly Thr Gly Ser Asp Ile Tyr Ile Trp 115 120 125 Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile 130 135 140 Thr Met His Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln 145 150 155 160 Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser 165 170 175 Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys 180 185 190 Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln 195 200 205 Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu 210 215 220 Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg 225 230 235 240 Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met 245 250 255 Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly 260 265 270 Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp 275 280 285 Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Ser Gly Ser 290 295 300 Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu 305 310 315 320 Asn Pro Gly Pro Ser Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu 325 330 335 Leu Leu Trp Val Pro Gly Ser Thr Gly Glu Val Gln Leu Val Glu Ser 340 345 350 Gly Gly Gly Glu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala 355 360 365 Ala Ser Arg Ser Ser Gly Ile Asp Val Met Gly Trp Tyr Arg Gln Ala 370 375 380 Pro Gly Lys Glu Arg Glu Leu Val Ala Glu Ile Ser Gly Val Gly Asp 385 390 395 400 Thr Asn Tyr Ala Ala Ser Leu Ala Asp Arg Phe Thr Val Ser Arg Asp 405 410 415 Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu 420 425 430 Asp Thr Ala Val Tyr Tyr Cys Asn Ala His Ser Phe Leu Asp Leu Val 435 440 445 Gly Ala Trp Gly Gln Gly Thr Leu Val Thr Val Lys Pro Gly Gly Gly 450 455 460 Gly Ser Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr 465 470 475 480 Phe Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr Thr Gly Met Leu 485 490 495 Glu Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe 500 505 510 Lys Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu Glu Gly 515 520 525 Val Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile Ser Pro 530 535 540 Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile Ile Pro His 545 550 555 560 Ala Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu Glu Gly Gly Arg 565 570 575 Met Ala Leu Ile Val Leu Gly Gly Val Ala Gly Leu Leu Leu Phe Ile 580 585 590 Gly Leu Gly Ile Phe Phe Lys Lys Lys Pro Leu Cys Leu Gln Arg Glu 595 600 605 Ala Lys Val Pro His Leu Pro Ala Asp Lys Ala Arg Gly Thr Gln Gly 610 615 620 Pro Glu Gln Gln His Leu Leu Ile Thr Ala Pro Ser Ser Ser Ser Ser Ser 625 630 635 640 Ser Leu Glu Ser Ser Ala Ser Ala Leu Asp Arg Arg Ala Pro Thr Arg 645 650 655 Asn Gln Pro Gln Ala Pro Gly Val Glu Ala Ser Gly Ala Gly Glu Ala 660 665 670 Arg Ala Ser Thr Gly Ser Ser Asp Ser Ser Pro Gly Gly His Gly Thr 675 680 685 Gln Val Asn Val Thr Cys Ile Val Asn Val Cys Ser Ser Ser Asp His 690 695 700 Ser Ser Gln Cys Ser Ser Gln Ala Ser Ser Thr Met Gly Asp Thr Asp 705 710 715 720 Ser Ser Pro Ser Glu Ser Pro Lys Asp Glu Gln Val Pro Phe Ser Lys 725 730 735 Glu Glu Cys Ala Phe Arg Ser Gln Leu Glu Thr Pro Glu Thr Leu Leu 740 745 750 Gly Ser Thr Glu Glu Lys Pro Leu Pro Leu Gly Val Pro Asp Ala Gly 755 760 765 Met Lys Pro Ser 770 <210> 61 <211> 776 <212> PRT <213> Artificial Sequence <220> <223> Made in Lab - anti-CD33 VHH DARIC.TNFR2 fusion protein <400> 61 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Gly Ser Ile Leu Trp His Glu Met Trp His Glu 20 25 30 Gly Leu Glu Glu Ala Ser Arg Leu Tyr Phe Gly Glu Arg Asn Val Lys 35 40 45 Gly Met Phe Glu Val Leu Glu Pro Leu His Ala Met Met Glu Arg Gly 50 55 60 Pro Gln Thr Leu Lys Glu Thr Ser Phe Asn Gln Ala Tyr Gly Arg Asp 65 70 75 80 Leu Met Glu Ala Gln Glu Trp Cys Arg Lys Tyr Met Lys Ser Gly Asn 85 90 95 Val Lys Asp Leu Leu Gln Ala Trp Asp Leu Tyr Tyr His Val Phe Arg 100 105 110 Arg Ile Ser Lys Ala Ser Ala Gly Thr Gly Ser Asp Ile Tyr Ile Trp 115 120 125 Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile 130 135 140 Thr Met His Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln 145 150 155 160 Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser 165 170 175 Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys 180 185 190 Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln 195 200 205 Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu 210 215 220 Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg 225 230 235 240 Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met 245 250 255 Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly 260 265 270 Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp 275 280 285 Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Ser Gly Ser 290 295 300 Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu 305 310 315 320 Asn Pro Gly Pro Ser Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu 325 330 335 Leu Leu Trp Val Pro Gly Ser Thr Gly Glu Val Gln Leu Val Glu Ser 340 345 350 Gly Gly Gly Glu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala 355 360 365 Ala Ser Gly Ser Thr Leu Asn Ile Asp His Ile Gly Trp Tyr Arg Gln 370 375 380 Ala Pro Gly Lys Glu Arg Glu Leu Val Gly Val Ile Ser Ser Gly Ala 385 390 395 400 Gly Pro Asn Tyr Ala Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg 405 410 415 Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Ser Ser Leu Arg Ala 420 425 430 Glu Asp Thr Ala Val Tyr Tyr Cys Asn Ala Trp Ile Asp Tyr Gly Ser 435 440 445 Gly Leu Pro Gln Asn Tyr Trp Gly Gly Gly Thr Leu Val Thr Val Lys 450 455 460 Pro Gly Gly Gly Gly Ser Gly Val Gln Val Glu Thr Ile Ser Pro Gly 465 470 475 480 Asp Gly Arg Thr Phe Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr 485 490 495 Thr Gly Met Leu Glu Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg 500 505 510 Asn Lys Pro Phe Lys Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly 515 520 525 Trp Glu Glu Gly Val Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu 530 535 540 Thr Ile Ser Pro Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile 545 550 555 560 Ile Pro Pro His Ala Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu 565 570 575 Glu Gly Gly Arg Met Ala Leu Ile Val Leu Gly Gly Val Ala Gly Leu 580 585 590 Leu Leu Phe Ile Gly Leu Gly Ile Phe Phe Lys Lys Lys Lys Pro Leu Cys 595 600 605 Leu Gln Arg Glu Ala Lys Val Pro His Leu Pro Ala Asp Lys Ala Arg 610 615 620 Gly Thr Gln Gly Pro Glu Gln Gln His Leu Leu Ile Thr Ala Pro Ser 625 630 635 640 Ser Ser Ser Ser Ser Leu Glu Ser Ser Ala Ser Ala Leu Asp Arg Arg 645 650 655 Ala Pro Thr Arg Asn Gln Pro Gln Ala Pro Gly Val Glu Ala Ser Gly 660 665 670 Ala Gly Glu Ala Arg Ala Ser Thr Gly Ser Ser Asp Ser Ser Pro Gly 675 680 685 Gly His Gly Thr Gln Val Asn Val Thr Cys Ile Val Asn Val Cys Ser 690 695 700 Ser Ser Asp His Ser Ser Gln Cys Ser Ser Gln Ala Ser Ser Thr Met 705 710 715 720 Gly Asp Thr Asp Ser Ser Pro Ser Glu Ser Pro Lys Asp Glu Gln Val 725 730 735 Pro Phe Ser Lys Glu Glu Cys Ala Phe Arg Ser Gln Leu Glu Thr Pro 740 745 750 Glu Thr Leu Leu Gly Ser Thr Glu Glu Lys Pro Leu Pro Leu Gly Val 755 760 765 Pro Asp Ala Gly Met Lys Pro Ser 770 775 <210> 62 <211> 372 <212> PRT <213> Artificial Sequence <220> <223> Made in Lab - anti-CD33 VHH chimeric antigen receptor <400> 62 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Gln Val Thr Leu Arg Glu Ser Gly Gly Gly Leu 20 25 30 Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Lys Gly Ser Gly Arg 35 40 45 Ala Ile Asn Thr Tyr Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys 50 55 60 Glu Arg Glu Phe Val Ala Ala Ile Ser Trp Asn Gly Gly His Thr Arg 65 70 75 80 Tyr Ala Asp Ser Val Gln Gly Arg Phe Ala Ile Ser Arg Asp Asn Ala 85 90 95 Asp Asn Thr Met Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr 100 105 110 Ala Val Tyr His Cys Ala Ala Tyr Ser Asp Tyr His Arg Ile Ala Thr 115 120 125 Met Glu Ala Asp Ala Asp Ser Trp Gly Gln Gly Thr Gln Val Thr Val 130 135 140 Ser Ser Ala Ala Ala Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro 145 150 155 160 Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys 165 170 175 Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala 180 185 190 Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu 195 200 205 Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys 210 215 220 Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr 225 230 235 240 Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly 245 250 255 Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala 260 265 270 Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg 275 280 285 Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu 290 295 300 Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn 305 310 315 320 Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met 325 330 335 Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly 340 345 350 Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala 355 360 365 Leu Pro Pro Arg 370 <210> 63 <211> 367 <212> PRT <213> Artificial Sequence <220> <223> Made in Lab - anti-CD33 VHH chimeric antigen receptor <400> 63 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Glu Val Gln Leu Val Glu Ser Gly Gly Gly Glu 20 25 30 Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg 35 40 45 Thr Phe Ser Gly Tyr Ile Met Gly Trp Phe Arg Gln Ala Pro Gly Lys 50 55 60 Glu Arg Glu Leu Val Ala Arg Ile Ser Gly Asn Asn Leu Ser Thr Glu 65 70 75 80 Tyr Ala Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala 85 90 95 Lys Asn Thr Leu Tyr Leu Gln Met Ser Leu Arg Ala Glu Asp Thr 100 105 110 Ala Val Tyr Tyr Cys Ala Ala Glu Tyr Asp Tyr Ser Ser Gly Asp Phe 115 120 125 Val Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Lys Pro Ala Ala Ala 130 135 140 Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala 145 150 155 160 Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly 165 170 175 Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile 180 185 190 Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val 195 200 205 Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe 210 215 220 Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly 225 230 235 240 Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg 245 250 255 Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln 260 265 270 Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp 275 280 285 Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro 290 295 300 Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp 305 310 315 320 Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg 325 330 335 Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr 340 345 350 Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 355 360 365 <210> 64 <211> 365 <212> PRT <213> Artificial Sequence <220> <223> Made in Lab - anti-CD33 VHH chimeric antigen receptor <400> 64 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Glu Val Gln Leu Val Glu Ser Gly Gly Gly Glu 20 25 30 Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser 35 40 45 Gly Phe Ser Ala Ser Leu Met Ser Trp His Arg Gln Ala Pro Gly Lys 50 55 60 Gln Arg Asp Leu Val Ala Ser Ile Thr Arg Asp Gly Arg Ala Asn Tyr 65 70 75 80 Val Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys 85 90 95 Asn Thr Leu Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala 100 105 110 Val Tyr Tyr Cys His Ala Tyr Ser Phe Asp Tyr Pro Ile Arg Ser Tyr 115 120 125 Trp Gly Gln Gly Thr Leu Val Thr Val Lys Pro Ala Ala Ala Thr Thr 130 135 140 Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln 145 150 155 160 Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala 165 170 175 Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala 180 185 190 Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr 195 200 205 Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln 210 215 220 Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser 225 230 235 240 Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys 245 250 255 Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln 260 265 270 Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu 275 280 285 Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg 290 295 300 Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met 305 310 315 320 Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly 325 330 335 Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp 340 345 350 Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 355 360 365 <210> 65 <211> 365 <212> PRT <213> Artificial Sequence <220> <223> Made in Lab - anti-CD33 VHH chimeric antigen receptor <400> 65 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Glu Val Gln Leu Val Glu Ser Gly Gly Gly Glu 20 25 30 Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe 35 40 45 Thr Phe Ser Ser Tyr Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys 50 55 60 Gly Arg Glu Trp Val Ala Ala Ile Thr Thr Ser Gly Asp Thr Thr Tyr 65 70 75 80 Tyr Ala Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala 85 90 95 Lys Asn Thr Val Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr 100 105 110 Ala Val Tyr Tyr Cys Ala Ala His Arg Gly Gly Gly Val Ile Asp Tyr 115 120 125 Trp Gly Gln Gly Thr Leu Val Thr Val Lys Pro Ala Ala Ala Thr Thr 130 135 140 Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln 145 150 155 160 Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala 165 170 175 Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala 180 185 190 Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr 195 200 205 Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln 210 215 220 Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser 225 230 235 240 Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys 245 250 255 Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln 260 265 270 Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu 275 280 285 Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg 290 295 300 Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met 305 310 315 320 Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly 325 330 335 Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp 340 345 350 Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 355 360 365 <210> 66 <211> 368 <212> PRT <213> Artificial Sequence <220> <223> Made in Lab - anti-CD33 VHH chimeric antigen receptor <400> 66 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Glu Val Gln Leu Val Glu Ser Gly Gly Gly Glu 20 25 30 Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser 35 40 45 Ile Phe Ser Ile Ser Ile Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys 50 55 60 Glu Arg Glu Leu Val Ala Ser Thr Thr Ser Ser Gly Thr Thr Asn Tyr 65 70 75 80 Val Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys 85 90 95 Asn Thr Leu Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala 100 105 110 Val Tyr Tyr Cys His Ala Tyr Ile Ala Thr Thr Thr Asp Arg Gly Tyr 115 120 125 Arg Gly Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Lys Pro Ala Ala 130 135 140 Ala Thr Thr Thr Pro Ala Pro Arg Pro Thr Pro Ala Pro Thr Ile 145 150 155 160 Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala 165 170 175 Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr 180 185 190 Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu 195 200 205 Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile 210 215 220 Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp 225 230 235 240 Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu 245 250 255 Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly 260 265 270 Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 275 280 285 Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys 290 295 300 Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys 305 310 315 320 Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg 325 330 335 Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala 340 345 350 Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 355 360 365 <210> 67 <211> 371 <212> PRT <213> Artificial Sequence <220> <223> Made in Lab - anti-CD33 VHH chimeric antigen receptor <400> 67 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu 20 25 30 Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser 35 40 45 Ile Ser Ser Tyr Asn Val Val Gly Trp Tyr Arg Gln Leu Ser Gly Asn 50 55 60 Glu Arg Gly Gly Arg Thr Met Val Ala Gln Ile Asn Ala Tyr Gly Asp 65 70 75 80 Thr Asn Tyr Ala Asn Ala Val Val Gly Arg Phe Thr Ile Ser Arg Asp 85 90 95 Asp Ala Lys Asn Thr Val Tyr Leu His Met Ser Asn Leu Lys Pro Glu 100 105 110 Asp Thr Gly Val Tyr Tyr Cys Asn Gly Gln Arg Met Leu Glu Asn Tyr 115 120 125 Thr Tyr Arg Asp Gln Ser Trp Gly Gly Gly Thr Gln Val Thr Val Lys 130 135 140 Pro Ala Ala Ala Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala 145 150 155 160 Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg 165 170 175 Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys 180 185 190 Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu 195 200 205 Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu 210 215 220 Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln 225 230 235 240 Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly 245 250 255 Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr 260 265 270 Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg 275 280 285 Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met 290 295 300 Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu 305 310 315 320 Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys 325 330 335 Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu 340 345 350 Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu 355 360 365 Pro Pro Arg 370 <210> 68 <211> 363 <212> PRT <213> Artificial Sequence <220> <223> Made in Lab - anti-CD33 VHH chimeric antigen receptor <400> 68 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu 20 25 30 Val Gln Ala Gly Gly Ser Leu Thr Leu Ser Cys Ala Ala Ser Arg Ser 35 40 45 Ser Gly Ile Asp Val Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu 50 55 60 Arg Glu Leu Val Ala Glu Ile Ser Gly Val Gly Asp Thr Asn Tyr Ala 65 70 75 80 Ala Ser Leu Ala Asp Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Asn 85 90 95 Thr Val Tyr Leu Gln Met Lys Asn Leu Lys Pro Glu Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Asn Ala His Ser Phe Leu Asp Leu Val Gly Ala Trp Gly 115 120 125 Gln Gly Thr Gln Val Thr Val Lys Pro Ala Ala Ala Thr Thr Thr Pro 130 135 140 Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu 145 150 155 160 Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His 165 170 175 Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu 180 185 190 Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr 195 200 205 Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe 210 215 220 Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg 225 230 235 240 Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser 245 250 255 Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr 260 265 270 Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys 275 280 285 Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn 290 295 300 Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu 305 310 315 320 Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly 325 330 335 His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr 340 345 350 Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 355 360 <210> 69 <211> 367 <212> PRT <213> Artificial Sequence <220> <223> Made in Lab - anti-CD33 VHH chimeric antigen receptor <400> 69 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Glu Val Gln Leu Val Gln Ser Gly Gly Gly Ser 20 25 30 Val Gln Val Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser 35 40 45 Thr Leu Asn Ile Asp His Ile Gly Trp Tyr Arg Gln Ala Pro Gly Lys 50 55 60 Glu Arg Glu Leu Val Gly Val Ile Ser Ser Gly Ala Gly Pro Asn Tyr 65 70 75 80 Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys 85 90 95 Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala 100 105 110 Val Tyr Asn Cys Asn Ala Trp Ile Asp Tyr Gly Ser Gly Leu Pro Gln 115 120 125 Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Lys Pro Ala Ala Ala 130 135 140 Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala 145 150 155 160 Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly 165 170 175 Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile 180 185 190 Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val 195 200 205 Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe 210 215 220 Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly 225 230 235 240 Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg 245 250 255 Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln 260 265 270 Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp 275 280 285 Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro 290 295 300 Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp 305 310 315 320 Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg 325 330 335 Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr 340 345 350 Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 355 360 365 <210> 70 <211> 363 <212> PRT <213> Artificial Sequence <220> <223> Made in Lab - anti-CD33 VHH chimeric antigen receptor <400> 70 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Glu Val Gln Leu Val Glu Ser Gly Gly Gly Glu 20 25 30 Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Arg Ser 35 40 45 Ser Gly Ile Asp Val Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu 50 55 60 Arg Glu Leu Val Ala Glu Ile Ser Gly Val Gly Asp Thr Asn Tyr Ala 65 70 75 80 Ala Ser Leu Ala Asp Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Asn 85 90 95 Thr Val Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Asn Ala His Ser Phe Leu Asp Leu Val Gly Ala Trp Gly 115 120 125 Gln Gly Thr Leu Val Thr Val Lys Pro Ala Ala Ala Thr Thr Thr Pro 130 135 140 Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu 145 150 155 160 Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His 165 170 175 Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu 180 185 190 Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr 195 200 205 Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe 210 215 220 Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg 225 230 235 240 Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser 245 250 255 Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr 260 265 270 Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys 275 280 285 Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn 290 295 300 Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu 305 310 315 320 Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly 325 330 335 His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr 340 345 350 Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 355 360 <210> 71 <211> 367 <212> PRT <213> Artificial Sequence <220> <223> Made in Lab - anti-CD33 VHH chimeric antigen receptor <400> 71 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Glu Val Gln Leu Val Glu Ser Gly Gly Gly Glu 20 25 30 Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser 35 40 45 Thr Leu Asn Ile Asp His Ile Gly Trp Tyr Arg Gln Ala Pro Gly Lys 50 55 60 Glu Arg Glu Leu Val Gly Val Ile Ser Ser Gly Ala Gly Pro Asn Tyr 65 70 75 80 Ala Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys 85 90 95 Asn Thr Val Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala 100 105 110 Val Tyr Tyr Cys Asn Ala Trp Ile Asp Tyr Gly Ser Gly Leu Pro Gln 115 120 125 Asn Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Lys Pro Ala Ala Ala 130 135 140 Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala 145 150 155 160 Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly 165 170 175 Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile 180 185 190 Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val 195 200 205 Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe 210 215 220 Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly 225 230 235 240 Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg 245 250 255 Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln 260 265 270 Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp 275 280 285 Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro 290 295 300 Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp 305 310 315 320 Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg 325 330 335 Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr 340 345 350 Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 355 360 365 <210> 72 <211> 369 <212> PRT <213> Artificial Sequence <220> <223> Made in Lab - anti-CD33 VHH chimeric antigen receptor <400> 72 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Gln Val Thr Leu Arg Glu Ser Gly Gly Gly Leu 20 25 30 Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Lys Gly Ser Gly Arg 35 40 45 Ala Ile Asn Thr Tyr Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys 50 55 60 Glu Arg Glu Phe Val Ala Ala Ile Ser Trp Asn Gly Gly His Thr Arg 65 70 75 80 Tyr Ala Asp Ser Val Gln Gly Arg Phe Ala Ile Ser Arg Asp Asn Ala 85 90 95 Asp Asn Thr Met Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr 100 105 110 Ala Val Tyr His Cys Ala Ala Tyr Ser Asp Tyr His Arg Ile Ala Thr 115 120 125 Met Glu Ala Asp Ala Asp Ser Trp Gly Gln Gly Thr Gln Val Thr Val 130 135 140 Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr 145 150 155 160 Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala 165 170 175 Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile 180 185 190 Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser 195 200 205 Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr 210 215 220 Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu 225 230 235 240 Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu 245 250 255 Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln 260 265 270 Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu 275 280 285 Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly 290 295 300 Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln 305 310 315 320 Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu 325 330 335 Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr 340 345 350 Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro 355 360 365 Arg <210> 73 <211> 364 <212> PRT <213> Artificial Sequence <220> <223> Made in Lab - anti-CD33 VHH chimeric antigen receptor <400> 73 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Glu Val Gln Leu Val Glu Ser Gly Gly Gly Glu 20 25 30 Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg 35 40 45 Thr Phe Ser Gly Tyr Ile Met Gly Trp Phe Arg Gln Ala Pro Gly Lys 50 55 60 Glu Arg Glu Leu Val Ala Arg Ile Ser Gly Asn Asn Leu Ser Thr Glu 65 70 75 80 Tyr Ala Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala 85 90 95 Lys Asn Thr Leu Tyr Leu Gln Met Ser Leu Arg Ala Glu Asp Thr 100 105 110 Ala Val Tyr Tyr Cys Ala Ala Glu Tyr Asp Tyr Ser Ser Gly Asp Phe 115 120 125 Val Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Lys Pro Thr Thr Thr 130 135 140 Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro 145 150 155 160 Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val 165 170 175 His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro 180 185 190 Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu 195 200 205 Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro 210 215 220 Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys 225 230 235 240 Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe 245 250 255 Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu 260 265 270 Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp 275 280 285 Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys 290 295 300 Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala 305 310 315 320 Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys 325 330 335 Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr 340 345 350 Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 355 360 <210> 74 <211> 362 <212> PRT <213> Artificial Sequence <220> <223> Made in Lab - anti-CD33 VHH chimeric antigen receptor <400> 74 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Glu Val Gln Leu Val Glu Ser Gly Gly Gly Glu 20 25 30 Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser 35 40 45 Gly Phe Ser Ala Ser Leu Met Ser Trp His Arg Gln Ala Pro Gly Lys 50 55 60 Gln Arg Asp Leu Val Ala Ser Ile Thr Arg Asp Gly Arg Ala Asn Tyr 65 70 75 80 Val Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys 85 90 95 Asn Thr Leu Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala 100 105 110 Val Tyr Tyr Cys His Ala Tyr Ser Phe Asp Tyr Pro Ile Arg Ser Tyr 115 120 125 Trp Gly Gln Gly Thr Leu Val Thr Val Lys Pro Thr Thr Thr Pro Ala 130 135 140 Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser 145 150 155 160 Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr 165 170 175 Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala 180 185 190 Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys 195 200 205 Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met 210 215 220 Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe 225 230 235 240 Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg 245 250 255 Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn 260 265 270 Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg 275 280 285 Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro 290 295 300 Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala 305 310 315 320 Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His 325 330 335 Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp 340 345 350 Ala Leu His Met Gln Ala Leu Pro Pro Arg 355 360 <210> 75 <211> 362 <212> PRT <213> Artificial Sequence <220> <223> Made in Lab - anti-CD33 VHH chimeric antigen receptor <400> 75 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Glu Val Gln Leu Val Glu Ser Gly Gly Gly Glu 20 25 30 Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe 35 40 45 Thr Phe Ser Ser Tyr Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys 50 55 60 Gly Arg Glu Trp Val Ala Ala Ile Thr Thr Ser Gly Asp Thr Thr Tyr 65 70 75 80 Tyr Ala Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala 85 90 95 Lys Asn Thr Val Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr 100 105 110 Ala Val Tyr Tyr Cys Ala Ala His Arg Gly Gly Gly Val Ile Asp Tyr 115 120 125 Trp Gly Gln Gly Thr Leu Val Thr Val Lys Pro Thr Thr Thr Pro Ala 130 135 140 Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser 145 150 155 160 Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr 165 170 175 Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala 180 185 190 Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys 195 200 205 Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met 210 215 220 Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe 225 230 235 240 Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg 245 250 255 Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn 260 265 270 Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg 275 280 285 Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro 290 295 300 Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala 305 310 315 320 Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His 325 330 335 Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp 340 345 350 Ala Leu His Met Gln Ala Leu Pro Pro Arg 355 360 <210> 76 <211> 365 <212> PRT <213> Artificial Sequence <220> <223> Made in Lab - anti-CD33 VHH chimeric antigen receptor <400> 76 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Glu Val Gln Leu Val Glu Ser Gly Gly Gly Glu 20 25 30 Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser 35 40 45 Ile Phe Ser Ile Ser Ile Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys 50 55 60 Glu Arg Glu Leu Val Ala Ser Thr Thr Ser Ser Gly Thr Thr Asn Tyr 65 70 75 80 Val Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys 85 90 95 Asn Thr Leu Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala 100 105 110 Val Tyr Tyr Cys His Ala Tyr Ile Ala Thr Thr Thr Asp Arg Gly Tyr 115 120 125 Arg Gly Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Lys Pro Thr Thr 130 135 140 Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln 145 150 155 160 Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala 165 170 175 Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala 180 185 190 Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr 195 200 205 Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln 210 215 220 Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser 225 230 235 240 Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys 245 250 255 Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln 260 265 270 Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu 275 280 285 Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg 290 295 300 Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met 305 310 315 320 Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly 325 330 335 Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp 340 345 350 Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 355 360 365 <210> 77 <211> 368 <212> PRT <213> Artificial Sequence <220> <223> Made in Lab - anti-CD33 VHH chimeric antigen receptor <400> 77 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu 20 25 30 Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser 35 40 45 Ile Ser Ser Tyr Asn Val Val Gly Trp Tyr Arg Gln Leu Ser Gly Asn 50 55 60 Glu Arg Gly Gly Arg Thr Met Val Ala Gln Ile Asn Ala Tyr Gly Asp 65 70 75 80 Thr Asn Tyr Ala Asn Ala Val Val Gly Arg Phe Thr Ile Ser Arg Asp 85 90 95 Asp Ala Lys Asn Thr Val Tyr Leu His Met Ser Asn Leu Lys Pro Glu 100 105 110 Asp Thr Gly Val Tyr Tyr Cys Asn Gly Gln Arg Met Leu Glu Asn Tyr 115 120 125 Thr Tyr Arg Asp Gln Ser Trp Gly Gly Gly Thr Gln Val Thr Val Lys 130 135 140 Pro Thr Thr Thr Pro Ala Pro Arg Pro Thr Pro Ala Pro Thr Ile 145 150 155 160 Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala 165 170 175 Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr 180 185 190 Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu 195 200 205 Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile 210 215 220 Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp 225 230 235 240 Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu 245 250 255 Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly 260 265 270 Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 275 280 285 Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys 290 295 300 Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys 305 310 315 320 Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg 325 330 335 Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala 340 345 350 Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 355 360 365 <210> 78 <211> 360 <212> PRT <213> Artificial Sequence <220> <223> Made in Lab - anti-CD33 VHH chimeric antigen receptor <400> 78 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu 20 25 30 Val Gln Ala Gly Gly Ser Leu Thr Leu Ser Cys Ala Ala Ser Arg Ser 35 40 45 Ser Gly Ile Asp Val Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu 50 55 60 Arg Glu Leu Val Ala Glu Ile Ser Gly Val Gly Asp Thr Asn Tyr Ala 65 70 75 80 Ala Ser Leu Ala Asp Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Asn 85 90 95 Thr Val Tyr Leu Gln Met Lys Asn Leu Lys Pro Glu Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Asn Ala His Ser Phe Leu Asp Leu Val Gly Ala Trp Gly 115 120 125 Gln Gly Thr Gln Val Thr Val Lys Pro Thr Thr Thr Pro Ala Pro Arg 130 135 140 Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg 145 150 155 160 Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly 165 170 175 Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr 180 185 190 Cys Gly Val Leu Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg 195 200 205 Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro 210 215 220 Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu 225 230 235 240 Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala 245 250 255 Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu 260 265 270 Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly 275 280 285 Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu 290 295 300 Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser 305 310 315 320 Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly 325 330 335 Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu 340 345 350 His Met Gln Ala Leu Pro Pro Arg 355 360 <210> 79 <211> 364 <212> PRT <213> Artificial Sequence <220> <223> Made in Lab - anti-CD33 VHH chimeric antigen receptor <400> 79 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Glu Val Gln Leu Val Gln Ser Gly Gly Gly Ser 20 25 30 Val Gln Val Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser 35 40 45 Thr Leu Asn Ile Asp His Ile Gly Trp Tyr Arg Gln Ala Pro Gly Lys 50 55 60 Glu Arg Glu Leu Val Gly Val Ile Ser Ser Gly Ala Gly Pro Asn Tyr 65 70 75 80 Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys 85 90 95 Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala 100 105 110 Val Tyr Asn Cys Asn Ala Trp Ile Asp Tyr Gly Ser Gly Leu Pro Gln 115 120 125 Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Lys Pro Thr Thr Thr 130 135 140 Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro 145 150 155 160 Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val 165 170 175 His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro 180 185 190 Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu 195 200 205 Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro 210 215 220 Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys 225 230 235 240 Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe 245 250 255 Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu 260 265 270 Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp 275 280 285 Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys 290 295 300 Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala 305 310 315 320 Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys 325 330 335 Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr 340 345 350 Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 355 360 <210> 80 <211> 360 <212> PRT <213> Artificial Sequence <220> <223> Made in Lab - anti-CD33 VHH chimeric antigen receptor <400> 80 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Glu Val Gln Leu Val Glu Ser Gly Gly Gly Glu 20 25 30 Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Arg Ser 35 40 45 Ser Gly Ile Asp Val Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu 50 55 60 Arg Glu Leu Val Ala Glu Ile Ser Gly Val Gly Asp Thr Asn Tyr Ala 65 70 75 80 Ala Ser Leu Ala Asp Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Asn 85 90 95 Thr Val Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Asn Ala His Ser Phe Leu Asp Leu Val Gly Ala Trp Gly 115 120 125 Gln Gly Thr Leu Val Thr Val Lys Pro Thr Thr Thr Pro Ala Pro Arg 130 135 140 Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg 145 150 155 160 Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly 165 170 175 Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr 180 185 190 Cys Gly Val Leu Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg 195 200 205 Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro 210 215 220 Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu 225 230 235 240 Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala 245 250 255 Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu 260 265 270 Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly 275 280 285 Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu 290 295 300 Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser 305 310 315 320 Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly 325 330 335 Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu 340 345 350 His Met Gln Ala Leu Pro Pro Arg 355 360 <210> 81 <211> 364 <212> PRT <213> Artificial Sequence <220> <223> Made in Lab - anti-CD33 VHH chimeric antigen receptor <400> 81 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Glu Val Gln Leu Val Glu Ser Gly Gly Gly Glu 20 25 30 Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser 35 40 45 Thr Leu Asn Ile Asp His Ile Gly Trp Tyr Arg Gln Ala Pro Gly Lys 50 55 60 Glu Arg Glu Leu Val Gly Val Ile Ser Ser Gly Ala Gly Pro Asn Tyr 65 70 75 80 Ala Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys 85 90 95 Asn Thr Val Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala 100 105 110 Val Tyr Tyr Cys Asn Ala Trp Ile Asp Tyr Gly Ser Gly Leu Pro Gln 115 120 125 Asn Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Lys Pro Thr Thr Thr 130 135 140 Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro 145 150 155 160 Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val 165 170 175 His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro 180 185 190 Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu 195 200 205 Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro 210 215 220 Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys 225 230 235 240 Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe 245 250 255 Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu 260 265 270 Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp 275 280 285 Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys 290 295 300 Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala 305 310 315 320 Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys 325 330 335 Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr 340 345 350 Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 355 360 <210> 82 <211> 301 <212> PRT <213> Artificial Sequence <220> <223> Made in Lab - anti-CD33 VHH DARIC signaling component <400> 82 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Gly Ser Ile Leu Trp His Glu Met Trp His Glu 20 25 30 Gly Leu Glu Glu Ala Ser Arg Leu Tyr Phe Gly Glu Arg Asn Val Lys 35 40 45 Gly Met Phe Glu Val Leu Glu Pro Leu His Ala Met Met Glu Arg Gly 50 55 60 Pro Gln Thr Leu Lys Glu Thr Ser Phe Asn Gln Ala Tyr Gly Arg Asp 65 70 75 80 Leu Met Glu Ala Gln Glu Trp Cys Arg Lys Tyr Met Lys Ser Gly Asn 85 90 95 Val Lys Asp Leu Leu Gln Ala Trp Asp Leu Tyr Tyr His Val Phe Arg 100 105 110 Arg Ile Ser Lys Ala Ser Ala Gly Thr Gly Ser Asp Ile Tyr Ile Trp 115 120 125 Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile 130 135 140 Thr Met His Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln 145 150 155 160 Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser 165 170 175 Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys 180 185 190 Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln 195 200 205 Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu 210 215 220 Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg 225 230 235 240 Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met 245 250 255 Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly 260 265 270 Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp 275 280 285 Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 290 295 300 <210> 83 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> Consensus Kozak sequence <400> 83 gccrccatgg 10 <210> 84 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Exemplary linker sequence <400> 84 Asp Gly Gly Gly Ser 1 5 <210> 85 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Exemplary linker sequence <400> 85 Thr Gly Glu Lys Pro 1 5 <210> 86 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Exemplary linker sequence <400> 86 Gly Gly Arg Arg One <210> 87 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Exemplary linker sequence <400> 87 Gly Gly Gly Gly Ser 1 5 <210> 88 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Exemplary linker sequence <400> 88 Glu Gly Lys Ser Ser Gly Ser Gly Ser Glu Ser Lys Val Asp 1 5 10 <210> 89 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Exemplary linker sequence <400> 89 Lys Glu Ser Gly Ser Val Ser Ser Glu Gln Leu Ala Gln Phe Arg Ser 1 5 10 15 Leu Asp <210> 90 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Exemplary linker sequence <400> 90 Gly Gly Arg Arg Gly Gly Gly Ser 1 5 <210> 91 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Exemplary linker sequence <400> 91 Leu Arg Gln Arg Asp Gly Glu Arg Pro 1 5 <210> 92 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Exemplary linker sequence <400> 92 Leu Arg Gln Lys Asp Gly Gly Gly Ser Glu Arg Pro 1 5 10 <210> 93 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Exemplary linker sequence <400> 93 Leu Arg Gln Lys Asp Gly Gly Gly Ser Gly Gly Gly Ser Glu Arg Pro 1 5 10 15 <210> 94 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Exemplary linker sequence <400> 94 Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr 1 5 10 15 Lys Gly <210> 95 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Cleavage sequence by TEV protease <220> <221> misc_feature <222> (2)..(3) <223> Xaa is any amino acid <220> <221> misc_feature <222> (5)..(5) <223> Xaa is any amino acid <220> <221> MISC_FEATURE <222> (7)..(7) <223> Xaa = Gly or Ser <400> 95 Glu Xaa Xaa Tyr Xaa Gln Xaa 1 5 <210> 96 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Cleavage sequence by TEV protease <400> 96 Glu Asn Leu Tyr Phe Gln Gly 1 5 <210> 97 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Cleavage sequence by TEV protease <400> 97 Glu Asn Leu Tyr Phe Gln Ser 1 5 <210> 98 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> Self-cleaving polypeptide comprising 2A site <400> 98 Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val 1 5 10 15 Glu Glu Asn Pro Gly Pro 20 <210> 99 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> Self-cleaving polypeptide comprising 2A site <400> 99 Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn 1 5 10 15 Pro Gly Pro <210> 100 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Self-cleaving polypeptide comprising 2A site <400> 100 Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro 1 5 10 <210> 101 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> Self-cleaving polypeptide comprising 2A site <400> 101 Gly Ser Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu 1 5 10 15 Glu Asn Pro Gly Pro 20 <210> 102 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Self-cleaving polypeptide comprising 2A site <400> 102 Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro 1 5 10 15 Gly Pro <210> 103 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Self-cleaving polypeptide comprising 2A site <400> 103 Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro Gly Pro 1 5 10 <210> 104 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> Self-cleaving polypeptide comprising 2A site <400> 104 Gly Ser Gly Gln Cys Thr Asn Tyr Ala Leu Leu Lys Leu Ala Gly Asp 1 5 10 15 Val Glu Ser Asn Pro Gly Pro 20 <210> 105 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Self-cleaving polypeptide comprising 2A site <400> 105 Gln Cys Thr Asn Tyr Ala Leu Leu Lys Leu Ala Gly Asp Val Glu Ser 1 5 10 15 Asn Pro Gly Pro 20 <210> 106 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Self-cleaving polypeptide comprising 2A site <400> 106 Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn Pro Gly Pro 1 5 10 <210> 107 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> Self-cleaving polypeptide comprising 2A site <400> 107 Gly Ser Gly Val Lys Gln Thr Leu Asn Phe Asp Leu Leu Lys Leu Ala 1 5 10 15 Gly Asp Val Glu Ser Asn Pro Gly Pro 20 25 <210> 108 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> Self-cleaving polypeptide comprising 2A site <400> 108 Val Lys Gln Thr Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val 1 5 10 15 Glu Ser Asn Pro Gly Pro 20 <210> 109 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Self-cleaving polypeptide comprising 2A site <400> 109 Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn Pro Gly Pro 1 5 10 <210> 110 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> Self-cleaving polypeptide comprising 2A site <400> 110 Leu Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn 1 5 10 15 Pro Gly Pro <210> 111 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> Self-cleaving polypeptide comprising 2A site <400> 111 Thr Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn 1 5 10 15 Pro Gly Pro <210> 112 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Self-cleaving polypeptide comprising 2A site <400> 112 Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn Pro Gly Pro 1 5 10 <210> 113 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Self-cleaving polypeptide comprising 2A site <400> 113 Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn Pro Gly 1 5 10 15 Pro <210> 114 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Self-cleaving polypeptide comprising 2A site <400> 114 Gln Leu Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val Glu Ser 1 5 10 15 Asn Pro Gly Pro 20 <210> 115 <211> 24 <212> PRT <213> Artificial Sequence <220> <223> Self-cleaving polypeptide comprising 2A site <400> 115 Ala Pro Val Lys Gln Thr Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly 1 5 10 15 Asp Val Glu Ser Asn Pro Gly Pro 20 <210> 116 <211> 40 <212> PRT <213> Artificial Sequence <220> <223> Self-cleaving polypeptide comprising 2A site <400> 116 Val Thr Glu Leu Leu Tyr Arg Met Lys Arg Ala Glu Thr Tyr Cys Pro 1 5 10 15 Arg Pro Leu Leu Ala Ile His Pro Thr Glu Ala Arg His Lys Gln Lys 20 25 30 Ile Val Ala Pro Val Lys Gln Thr 35 40 <210> 117 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Self-cleaving polypeptide comprising 2A site <400> 117 Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn Pro 1 5 10 15 Gly Pro <210> 118 <211> 40 <212> PRT <213> Artificial Sequence <220> <223> Self-cleaving polypeptide comprising 2A site <400> 118 Leu Leu Ala Ile His Pro Thr Glu Ala Arg His Lys Gln Lys Ile Val 1 5 10 15 Ala Pro Val Lys Gln Thr Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly 20 25 30 Asp Val Glu Ser Asn Pro Gly Pro 35 40 <210> 119 <211> 33 <212> PRT <213> Artificial Sequence <220> <223> Self-cleaving polypeptide comprising 2A site <400> 119 Glu Ala Arg His Lys Gln Lys Ile Val Ala Pro Val Lys Gln Thr Leu 1 5 10 15 Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn Pro Gly 20 25 30 Pro

Claims (94)

As a non-natural cell,
(a) an FRB multimerization domain polypeptide or variant thereof; CD8α transmembrane domain or CD4 transmembrane domain; CD137 costimulatory domain; and a first polypeptide comprising a CD3ζ primary signal transduction domain and
(b) an anti-CD33 VHH antibody having the amino acid sequence set forth in any one of SEQ ID NOs: 2-21; FKBP multimerization domain polypeptide or a variant thereof; and a second polypeptide comprising a CD4 transmembrane domain or a CD8α transmembrane domain;
A bridging factor is associated with and disposed between the first and second multimerization domains to promote formation of a polypeptide complex on the surface of the non-native cell. The non-naturally occurring cell of claim 1 , wherein the FKBP multimerization domain is FKBP12. The non-naturally occurring cell according to claim 1 or 2, wherein the FRB polypeptide is FRB T2098L. The non-naturally occurring cell according to any one of claims 1 to 3, wherein the bridging factor is selected from the group consisting of: AP21967, sirolimus, everolimus, novolimus, pimecrolimus, lidaforoli mousse, tacrolimus, temsirolimus, umirolimus and zotarolimus. 5. The method of any one of claims 1 to 4, wherein the first polypeptide comprises a signal peptide, a CD8α transmembrane domain; CD137 costimulatory domain; and a CD3ζ primary signaling domain. 6. The non-naturally occurring cell according to any one of claims 1 to 5, wherein the second polypeptide comprises a signal peptide and a CD4 transmembrane domain. 7. The non-naturally occurring cell according to any one of claims 1 to 6, wherein the second polypeptide comprises a costimulatory domain. 8. The non-natural cell of claim 7, wherein the costimulatory domain of the second polypeptide is selected from a costimulatory molecule selected from the group consisting of: Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, caspase recruitment domain family member 11 (CARD11), CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD94, CD134 (OX40), CD137 (4-1BB) ), CD278 (ICOS), DNAX-activating protein 10 (DAP10), T cell lineage member 1 (LAT), leukocyte protein of 76 kD containing SH2 domain (SLP76), T cell receptor-associated transmembrane adapter 1 (TRAT1), TNFR2 , TNFRS14, TNFRS18, TNRFS25, and the zeta chain of T cell receptor associated protein kinase 70 (ZAP70). 9. The non-naturally occurring cell of claim 7 or 8, wherein the costimulatory domain of the second polypeptide is a costimulatory domain isolated from OX40 or TNFR2. 10. The non-naturally occurring cell according to any one of claims 1 to 9, wherein the second polypeptide comprises a sequence set forth in any one of SEQ ID NOs: 22-31. A non-naturally occurring cell comprising a polypeptide complex, the non-naturally occurring cell comprising:
(a) an FRB multimerization domain polypeptide or variant thereof; CD8α transmembrane domain or CD4 transmembrane domain; CD137 costimulatory domain; and a first polypeptide comprising a CD3ζ primary signal transduction domain;
(b) an anti-CD33 VHH antibody having the amino acid sequence set forth in any one of SEQ ID NOs: 2-21; FKBP multimerization domain polypeptide or a variant thereof; and a second polypeptide comprising a CD4 transmembrane domain or a CD8α transmembrane domain; and
(c) a polypeptide complex comprising a bridging factor disposed between and associated with the multimerization domains of the first and second polypeptides. The non-naturally occurring cell of claim 11 , wherein the FKBP multimerization domain is FKBP12. The non-naturally occurring cell according to claim 11 or 12, wherein the FRB polypeptide is FRB T2098L. 14. A non-naturally occurring cell according to any one of claims 11 to 13, wherein the bridging factor is selected from the group consisting of: AP21967, sirolimus, everolimus, novolimus, pimecrolimus, ridaforoli mousse, tacrolimus, temsirolimus, umirolimus and zotarolimus. 15. The method of any one of claims 11-14, wherein the first polypeptide comprises a signal peptide, a CD8α transmembrane domain; CD137 costimulatory domain; and a CD3ζ primary signaling domain. 16. The non-native cell of any one of claims 11-15, wherein the second polypeptide comprises a signal peptide and a CD4 transmembrane domain. 17. The non-naturally occurring cell of any one of claims 11-16, wherein the second polypeptide comprises a costimulatory domain. 18. The non-natural cell of claim 17, wherein the costimulatory domain of the second polypeptide is selected from a costimulatory molecule selected from the group consisting of: Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, caspase recruitment domain family member 11 (CARD11), CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD94, CD134 (OX40), CD137 (4-1BB) ), CD278 (ICOS), DNAX-activating protein 10 (DAP10), T cell lineage member 1 (LAT), leukocyte protein of 76 kD containing SH2 domain (SLP76), T cell receptor-associated transmembrane adapter 1 (TRAT1), TNFR2 , TNFRS14, TNFRS18, TNRFS25, and the zeta chain of T cell receptor associated protein kinase 70 (ZAP70). 19. The non-naturally occurring cell of claim 17 or 18, wherein the costimulatory domain of the second polypeptide is a costimulatory domain isolated from OX40 or TNFR2. 20. The non-naturally occurring cell of any one of claims 11-19, wherein the second polypeptide comprises a sequence set forth in any one of SEQ ID NOs: 22-31. 21. The non-naturally occurring cell according to any one of claims 1 to 20, wherein the cell is a hematopoietic cell. 22. The non-naturally occurring cell of any one of claims 1-21, wherein the cell is a T cell, an αβ T cell, or a γδ T cell. 23. The non-naturally occurring cell of any one of claims 1-22, wherein the cell is a CD3 ⁺ , CD4 ⁺ , and/or CD8 ⁺ cell. 24. The non-naturally occurring cell of any one of claims 1-23, wherein the cell is an immune effector cell. 25. The non-naturally occurring cell of any one of claims 1-24, wherein the cell is a cytotoxic T lymphocyte (CTL), a tumor infiltrating lymphocyte (TIL), or a helper T cell. 26. The non-naturally occurring cell according to any one of claims 1 to 25, wherein the cell is a natural killer (NK) cell or a natural killer T (NKT) cell. 27. The method of any one of claims 1-26, wherein the source of cells is peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue derived from the site of infection, ascites, pleural effusion, spleen tissue, or a tumor. Phosphorus, non-natural cells. 28. The non-naturally occurring cell of any one of claims 1-27, wherein the FRB multimerization domain and the FKBP multimerization domain localize extracellularly when the first polypeptide and the second polypeptide are expressed. A fusion polypeptide comprising:
(a) an FRB multimerization domain polypeptide or variant thereof; CD8α transmembrane domain or CD4 transmembrane domain; CD137 costimulatory domain; and a first polypeptide comprising a CD3ζ primary signal transduction domain;
(b) a polypeptide cleavage signal; and
(c) an anti-CD33 VHH antibody having the amino acid sequence set forth in any one of SEQ ID NOs: 2-21; FKBP multimerization domain polypeptide or a variant thereof; and a second polypeptide comprising a CD4 transmembrane domain or a CD8α transmembrane domain; 30. The fusion polypeptide of claim 29, wherein the FKBP multimerization domain is FKBP12. 31. The fusion polypeptide of claim 29 or 30, wherein the FRB polypeptide is FRB T2098L. 32. The fusion polypeptide according to any one of claims 29 to 31, wherein the bridging factor is selected from the group consisting of: AP21967, sirolimus, everolimus, novolimus, pimecrolimus, ridaforolimus , tacrolimus, temsirolimus, umirolimus and zotarolimus. 33. The method of any one of claims 29-32, wherein the first polypeptide comprises a signal peptide, a CD8α transmembrane domain; CD137 costimulatory domain; and a CD3ζ primary signaling domain. 34. The fusion polypeptide of any one of claims 29-33, wherein the second polypeptide comprises a signal peptide and a CD4 transmembrane domain. 35. The non-naturally occurring cell of any one of claims 29-34, wherein the fusion polypeptide comprises a sequence set forth in any one of SEQ ID NOs: 32-41. 36. The fusion polypeptide of any one of claims 29-35, wherein the second polypeptide comprises a costimulatory domain. 37. The fusion polypeptide of claim 36, wherein the costimulatory domain of the second polypeptide is selected from a costimulatory molecule selected from the group consisting of: Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7. , TLR8, TLR9, TLR10, caspase recruitment domain family member 11 (CARD11), CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD94, CD134 (OX40), CD137 (4-1BB) , CD278 (ICOS), DNAX-activating protein 10 (DAP10), T cell lineage member 1 (LAT), leukocyte protein of 76 kD containing SH2 domain (SLP76), T cell receptor-associated transmembrane adapter 1 (TRAT1), TNFR2, TNFRS14, TNFRS18, TNRFS25, and the zeta chain of T cell receptor associated protein kinase 70 (ZAP70). 38. The fusion polypeptide of claim 36 or 37, wherein the costimulatory domain of the second polypeptide is a costimulatory domain isolated from OX40 or TNFR2. 39. The fusion polypeptide of any one of claims 29-38, wherein the polypeptide cleavage signal is a viral self-cleaving polypeptide. 40. The fusion polypeptide of any one of claims 29-39, wherein the polypeptide cleavage signal is a viral self-cleaving 2A polypeptide. 41. The fusion polypeptide according to any one of claims 29 to 40, wherein the polypeptide cleavage signal is a viral self-cleaving polypeptide selected from the group consisting of: foot-and-mouth disease virus (FMDV) (F2A) peptide, equine rhinitis A virus (ERAV) (E2A) peptide, Torcea acigna virus (TaV) (T2A) peptide, porcine Tescovirus-1 (PTV-1) (P2A) peptide, tailovirus 2A peptide, and encephalomyocarditis virus 2A peptide. 42. The non-naturally occurring cell of any one of claims 29-41, wherein the fusion polypeptide comprises a sequence set forth in any one of SEQ ID NOs: 42-61. 43. The fusion polypeptide of any one of claims 29-42, wherein the FRB multimerization domain and the FKBP multimerization domain localize extracellularly when the first polypeptide and the second polypeptide are expressed. A polypeptide complex comprising:
(a) an FRB multimerization domain polypeptide or variant thereof; CD8α transmembrane domain or CD4 transmembrane domain; CD137 costimulatory domain; and a first polypeptide comprising a CD3ζ primary signal transduction domain;
(b) an anti-CD33 VHH antibody having the amino acid sequence set forth in any one of SEQ ID NOs: 2-21; FKBP multimerization domain polypeptide or a variant thereof; and a second polypeptide comprising a CD4 transmembrane domain or a CD8α transmembrane domain; and
(c) a polypeptide complex comprising a bridging factor disposed between and associated with the multimerization domains of the first and second polypeptides. 45. The polypeptide complex of claim 44, wherein the FKBP multimerization domain is FKBP12. 46. The polypeptide complex of claim 44 or 45, wherein the FRB polypeptide is FRB T2098L. 47. The polypeptide complex according to any one of claims 44 to 46, wherein the bridging factor is selected from the group consisting of: AP21967, sirolimus, everolimus, novolimus, pimecrolimus, ridaforolimus , tacrolimus, temsirolimus, umirolimus and zotarolimus. 48. The method of any one of claims 44-47, wherein the first polypeptide comprises a CD8α transmembrane domain; CD137 costimulatory domain; and a CD3ζ primary signal transduction domain. 49. The polypeptide complex of any one of claims 44-48, wherein the second polypeptide comprises a CD4 transmembrane domain. 50. The polypeptide complex of any one of claims 44-49, wherein the second polypeptide comprises a costimulatory domain. 51. The polypeptide complex of claim 50, wherein the costimulatory domain of the second polypeptide is selected from a costimulatory molecule selected from the group consisting of: Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7. , TLR8, TLR9, TLR10, caspase recruitment domain family member 11 (CARD11), CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD94, CD134 (OX40), CD137 (4-1BB) , CD278 (ICOS), DNAX-activating protein 10 (DAP10), T cell lineage member 1 (LAT), leukocyte protein of 76 kD containing SH2 domain (SLP76), T cell receptor-associated transmembrane adapter 1 (TRAT1), TNFR2, TNFRS14, TNFRS18, TNRFS25, and the zeta chain of T cell receptor associated protein kinase 70 (ZAP70). 52. The polypeptide complex of claim 50 or 51, wherein the costimulatory domain of the second polypeptide is a costimulatory domain isolated from OX40 or TNFR2. 53. The polypeptide complex of any one of claims 44-52, wherein the cell is a hematopoietic cell. 54. The polypeptide complex of any one of claims 44-53, wherein the cell is a T cell, an αβ T cell, or a γδ T cell. 55. The polypeptide complex of any one of claims 44-54, wherein the cell is a CD3 ⁺ , CD4 ⁺ , and/or CD8 ⁺ cell. 56. The polypeptide complex of any one of claims 44-55, wherein the cell is an immune effector cell. 57. The polypeptide complex of any one of claims 44-56, wherein the cell is a cytotoxic T lymphocyte (CTL), a tumor infiltrating lymphocyte (TIL), or a helper T cell. 58. The polypeptide complex of any one of claims 44-57, wherein the cell is a natural killer (NK) cell or a natural killer T (NKT) cell. 59. The method of any one of claims 44-58, wherein the source of cells is peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue derived from the site of infection, ascites, pleural effusion, spleen tissue, or a tumor. phosphorus, a polypeptide complex. 60. The polypeptide complex of any one of claims 44-59, wherein the FRB multimerization domain and the FKBP multimerization domain localize extracellularly when the first polypeptide and the second polypeptide are expressed. A chimeric antigen receptor (CAR) comprising:
a) an anti-CD33 VHH antibody having the amino acid sequence set forth in any one of SEQ ID NOs: 2-21;
b) a hinge domain;
c) a transmembrane domain;
d) one or more intracellular costimulatory signaling domains; and/or
e) primary signaling domain 62. The CAR of claim 61, wherein the CAR comprises in the 5' to 3' direction:
a) an anti-CD33 VHH antibody having the amino acid sequence set forth in any one of SEQ ID NOs: 2-21;
b) a hinge domain;
c) a transmembrane domain;
d) one or more intracellular costimulatory signaling domains; and/or
e) primary signaling domain 63. The method of claim 61 or 62, wherein the hinge domain and the transmembrane domain are CD8α, CD27, CD28, CD33, CD37, CD45, CD64, CD71, CD80, CD86, CD 134, CD137, CD152, CD154, AMN, and PD1. isolated from, CAR. 64. The CAR of any one of claims 61-63, wherein the one or more costimulatory domains is isolated from a costimulatory molecule selected from the group consisting of: CD28, CD134, CD137, and CD278. 65. The CAR of any one of claims 61-64, wherein the CAR comprises a CD8α signal peptide, a CD8α hinge and transmembrane domain, a CD134 costimulatory domain, and a CD3ζ primary signaling domain. A CAR comprising the amino acid sequence set forth in any one of SEQ ID NOs: 62-81. A polynucleotide encoding the first or second polypeptide of any one of claims 1-28, the fusion polypeptide of any one of claims 29-43, or the CAR of any one of claims 61-66 . 67. A cDNA encoding the first or second polypeptide of any one of claims 1-28, the fusion polypeptide of any one of claims 29-43, or the CAR of any one of claims 61-66. 67. An RNA encoding the first or second polypeptide of any one of claims 1-28, the fusion polypeptide of any one of claims 29-43, or the CAR of any one of claims 61-66. 70. A vector comprising the polynucleotide of any one of claims 67 to 69. 71. The vector of claim 70, wherein the vector is an expression vector. 71. The vector of claim 70, wherein the vector is a transposon. 73. The vector of claim 72, wherein the vector is a piggyBAC transposon or a sleeping beauty transposon. 71. The vector of claim 70, wherein the vector is a viral vector. 75. The vector of claim 74, wherein the vector is an adenoviral vector, an adeno-associated virus (AAV) vector, a herpes virus vector, a vaccinia virus vector, or a retroviral vector. 76. The vector of claim 75, wherein the retroviral vector is a lentiviral vector. 77. The vector of claim 76, wherein the lentiviral vector is selected from the group consisting of: human immunodeficiency virus 1 (HIV-1); Human Immunodeficiency Virus 2 (HIV-2), Visna-Maedi Virus (VMV) Virus; caprin arthritis-encephalitis virus (CAEV); Equine Infectious Anemia Virus (EIAV); Feline Immunodeficiency Virus (FIV); bovine immunodeficiency virus (BIV); and simian immunodeficiency virus (SIV). 67. A cell comprising the first or second polypeptide of any one of claims 1-28, the fusion polypeptide of any one of claims 29-43, or the CAR of any one of claims 61-66. 79. The cell of claim 78, wherein the cell is a hematopoietic cell. 80. The cell of claim 78 or 79, wherein the cell is an immune effector cell. 81. The cell of any one of claims 78-80, wherein the cell is a T cell, an αβ T cell, or a γδ T cell. 82. The cell of any one of claims 78-81, wherein the cell expresses CD3 ^{+ ,} CD4 ⁺ , CD8 ⁺ , or a combination thereof. 83. The cell of any one of claims 78-82, wherein the cell is a cytotoxic T lymphocyte (CTL), a tumor infiltrating lymphocyte (TIL), or a helper T cell. 84. The cell of any one of claims 78-83, wherein the cell is a natural killer (NK) cell or a natural killer T (NKT) cell. 89. A composition comprising a cell according to any one of claims 1-28 and 78-84. 89. A composition comprising a physiologically acceptable carrier and a cell according to any one of claims 1-28 and 78-84. 87. A method of treating a subject in need thereof, comprising administering to the subject an effective amount of the composition of claim 85 or 86. 87. A method of treating, preventing, or ameliorating at least one symptom of cancer, infectious disease, autoimmune disease, inflammatory disease, and immunodeficiency, or a condition related thereto, wherein an effective amount of the composition of claim 85 or 86 is administered to a subject A method comprising the step of 87. A method of treating solid cancer comprising administering to a subject an effective amount of the composition of claim 85 or 86. 91. The method of claim 89, wherein the solid cancer is selected from the group consisting of lung cancer, squamous cell carcinoma, colorectal cancer, pancreatic cancer, breast cancer, thyroid cancer, bladder cancer, cervical cancer, esophageal cancer, ovarian cancer, stomach cancer, endometrial cancer, or brain cancer. 91. The method of claim 89 or 90, wherein the solid cancer is non-small cell lung carcinoma, head and neck squamous cell carcinoma, colorectal cancer, pancreatic cancer, breast cancer, thyroid cancer, bladder cancer, cervical cancer, esophageal cancer, ovarian cancer, gastric cancer, endometrial cancer, glioma , neuroblastoma, or oligodendroglioma. 87. A method of treating hematologic cancer comprising administering to a subject an effective amount of the composition of claim 85 or 86. 93. The method of claim 92, wherein the hematologic cancer is leukemia, lymphoma, or multiple myeloma. 93. The method of claim 92, wherein the hematologic cancer is acute myeloid leukemia (AML).

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